Polymerases for analyzing or typing polymorphic nucleic acid fragments and uses thereof

ABSTRACT

The present invention provides methods for use in identifying, analyzing and typing polymorphic DNA fragments, particularly minisatellite, microsatellite or STR DNA fragments. In particular, the invention provides methods using DNA polymerases, more particularly thermostable DNA polymerases, and most particularly Thermotoga polymerases or mutants or derivatives thereof, whereby minisatellite, microsatellite or STRDNA molecules may be amplified and analyzed for polymorphisms. The invention also relates to polymerases having reduced, substantially reduced or eliminated ability to add non-template 3′ nucleotides to a synthesized nucleic acid molecule. In accordance with the invention, such reduction or elimination may be accomplished by modifying or mutating the desired polymerase.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/037,393, filed Feb. 7, 1997, and to the U.S. ProvisionalApplication of Deb K. Chatterjee, Joseph Solus and Shuwei Yang, entitled“Polymerases for Analyzing or Typing Polymorphic Nucleic Acid Fragmentsand Uses Thereof,” filed Jan. 6, 1998, the disclosures of which arefully incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention is in the field of molecular and cellularbiology. The invention relates to compositions and methods for use inanalyzing and typing polymorphic regions of DNA. More particularly, theinvention is directed to compositions of polymerases (preferably DNApolymerases and most preferably thermostable DNA polymerases), andmethods using these compositions, whereby polymorphic, minisatellite,microsatellite or STRDNA fragments may be amplified and analyzed. Thecompositions and methods of the present invention are useful in avariety of techniques employing DNA amplification and polymorphismanalysis, including medical genetic, forensic, and plant breedingapplications.

[0003] The present invention also relates to polymerases having reduced,substantially reduced or eliminated ability to add one or morenon-templated nucleotides to the 3′terminus of a synthesized nucleicacid molecule. Preferably, the polymerases of the invention arethermostable or mesophilic polymerases. Specifically, the polymerases ofthe present invention (e.g., DNA or RNA polymerases) have been mutatedor modified to reduce, substantially reduce or eliminate such activity(compared to the unmodified, unmutated, or wild type polymerase),thereby providing a polymerase which synthesizes nucleic acid moleculeshaving little or no non-templated 3′terminal nucleotides. Suchpolymerases thus have enhanced or greater ability to produce a doublestranded nucleic acid molecule having blunt ended termini which mayfacilitate cloning of such molecules. The present invention also relatesto cloning and expression of the polymerases of the invention, tonucleic acid molecules containing the cloned genes, and to host cellswhich express said genes. The polymerases of the present invention maybe used in DNA sequencing, amplification, nucleic acid synthesis, andpolymorphism analysis.

[0004] The invention also relates to polymerases of the invention whichhave one or more additional mutations or modifications. Such mutationsor modifications include those which (1) substantially reduce 3′→5′exonuclease activity; and/or (2) substantially reduce 5′→3′ exonucleaseactivity. The polymerases of the invention can have one or more of theseproperties. These polymerases may also be used in nucleic acid analysisincluding but not limited to DNA sequencing, amplification, nucleic acidsynthesis, and polymorphism analysis.

BACKGROUND OF THE INVENTION

[0005] DNA Structure

[0006] The genetic framework (i.e., the genome) of an organism isencoded in the double-stranded sequence of nucleotide bases in thedeoxyribonucleic acid (DNA) which is contained in the somatic and germcells of the organism. The genetic content of a particular segment ofDNA, or gene, is only manifested upon production of the protein whichthe gene ultimately encodes. There are additional sequences in thegenome that do not encode a protein (i.e., “noncoding” regions) whichmay serve a structural, regulatory, or unknown function. Thus, thegenome of an organism or cell is the complete collection ofprotein-encoding genes together with intervening noncoding DNAsequences. Importantly, each somatic cell of a multicellular organismcontains the full complement of genonic DNA of the organism, except incases of focal infections or cancers, where one or more xenogeneic DNAsequences may be inserted into the genomic DNA of specific cells and notinto other, non-infected, cells in the organism.

[0007] Minisatellite and Microsatellite DNA

[0008] Interspersed throughout the genomic DNA of most eukaryoticorganisms are short stretches of polymorphic repetitive nucleotidesequences known as “minisatellite DNA” sequences or fragments (Jeffreys,A. J., et al., Nature 314:67-73 (1985)). These repeating sequences oftenappear in tandem and in variable numbers within the genome, and they arethus sometimes referred to as “short tandem repeats” (“STRs”) or“variable numbers of tandem repeats” (“VNTRs”) (see U.S. Pat. No.5,075,217; Nakamura et al., Science 235:1616-1622 (1987)). Typically,however, minisatellite repeat units are about 9 to 60 bases in length(Nakamura et al., Science 235:1616-1622 (1987); Weber and May, Am. JHum. Genet. 44:388-396 (1989)) which are repeated in tandem about 20-50times (Watson, J. D., et al., eds., Recombinant DNA, 2nd ed., New York:Scientific American Books, p. 146 (1992)). Other short, simple sequenceswhich are analogous to minisatellite DNAs, termed “microsatellite DNAs”(Litt, M., and Luty, J. A., Am. J. Hum. Genet 44:397-401 (1989); Weberand May, Am. J. Hum. Genet. 44:388-396 (1989)), are usually about 1-6bases in repeat unit length and thus give rise to monomeric (Economou,E. T., et al., Proc. Natl. Acad Sci. USA 87:2951-2954 (1990)), dimeric,trimeric, quatrameric, pentameric or hexameric repeat units (Litt, M.,and Luty, J. A., Am. J. Hum. Genet 44:397-401 (1989); Weber and May, Am.J. Hum. Genet. 44:388-396 (1989)). The most prevalent of these highlypolymorphic microsatellite sequences in the human genome is thedinucleotide repeat (dC-dA)_(n)•(dG-dT)_(n) (where n is the number ofrepetitions in a given stretch of nucleotides), which is present in acopy number of about 50,000-100,000 (Tautz, D., and Renz, M., Nucl.Acids Res. 12:4127-4138 (1984); Dib, C., et al., Nature 360:152-154(1996)), although the existence of a variety of analogous repeatsequences in the genomes of evolutionarily diverse eukaryotes has beenreported (Hamada, H., et al., Proc. Natl. Acad. Sci. USA 79:6465-6469(1982)).

[0009] The actual in vivo function of minisatellite and microsatellitesequences is unknown. However, because these tandemly repeated sequencesare dispersed throughout the genome of most eukaryotes, exhibit sizepolymorphism, and are often heterozygous (Weber, J. L., Genomics7:524-530 (1990)), they have been explored as potential genetic markersin assays attempting to distinguish closely related individuals, and inforensic and paternity testing (see, e.g., U.S. Pat. No. 5,075,217;Jeffreys, A. J., et al., Nature 332:278-281 (1988)). The finding thatmutations often are observed in microsatellite DNA regions in cancercells (Loeb, L. A., Cancer Res. 54:5059-5063 (1994)), potentiallylinking genomic instability to the carcinogenic process and providinguseful genetic markers of cancer, lends additional significance tomethods facilitating the rapid analysis and genotyping of polymorphismsin these genomic DNA regions.

[0010] Methods of Genotyping Minisatellite or STR DNA Sequences

[0011] To analyze minisatellite, microsatellite or STR DNA sequencepolymorphisms, a variety of molecular biological techniques have beenemployed. These techniques include restriction fragment lengthpolymorphism (RFLP) or “DNA fingerprinting” analysis (Wong, Z., et al.,Nucl. Acids Res. 14:4605-4616 (1986); Wong, Z., et al., Ann. Hum. Genet51:269-288 (1987); Jeffreys, A. J., et al., Nature 332:278-281 (1988);U.S. Pat. Nos. 5,175,082; 5,413,908; 5,459,039; and 5,556,955). Far morecommonly employed for STR genotyping than RFLP and hybridization,however, are amplification-based methods, such as those relying on thepolymerase chain reaction (PCR) method invented by Mullis and colleagues(see U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159). These methodsuse “primer” sequences which are complementary to opposing regionsflanking the polymorphic DNA sequence to be amplified from the sample ofgenomic DNA to be analyzed. These primers are added to the DNA targetsample, along with excess deoxynucleotides and a DNA polymerase (e.g.,Taq polymerase; see below), and the primers bind to their target viabase-specific binding interactions (i.e., adenine binds to thymine,cytosine to guanine). By repeatedly passing the reaction mixture throughcycles of increasing and decreasing temperatures (to allow dissociationof the two DNA strands on the target sequence, synthesis ofcomplementary copies of each strand by the polymerase, and re-annealingof the new complementary strands), the copy number of the minisatelliteor STR sequence of DNA may be rapidly increased, and detected by sizeseparation methods such as gel electrophoresis.

[0012] PCR and related amplification approaches have been used inattempts to develop methods for typing and analyzing STRs orminisatellite regions. For example, PCR has been employed to analyzepolymorphisms in microsatellite sequences from different individuals,including (dC-dA)n.(dG-dT)n (Weber, J. L, and May, P. E., Am. J. Hum.Genet. 44:388-396 (1989); Weber, J. L., Genomics 7:524-530 (1990); U.S.Pat. Nos. 5,075,217; 5,369,004; and 5,468,613). Similar methods havebeen applied to a variety of medical and forensic samples to perform DNAtyping and to detect polymorphisms between individual samples (U.S. Pat.Nos. 5,306,616; 5,364,759; 5,378,602; and 5,468,610).

[0013] in vitro Use of DNA Polymerases

[0014] The above-described amplification-based techniques require theuse of DNA polymerases, which catalyze the addition of deoxynucleosidetriphosphate (dNTP) bases into the newly forming DNA strands. Togetherwith other enzymes (e.g., helicases, ligases and ATPases), the DNApolymerases ensure rapid and relatively faithful replication of DNA inpreparation for proliferation in vivo in prokaryotes, eukaryotes andviruses.

[0015] DNA polymerases synthesize the formation of DNA molecules whichare complementary to a DNA template. Upon hybridization of a primer tothe single-stranded DNA template, polymerases synthesize DNA in the 5′to3′ direction, successively adding nucleotides to the 3′-hydroxyl groupof the growing strand. Thus, in the presence of deoxyribonucleosidetriphosphates (dNTPs) and a primer, a new DNA molecule, complementary tothe single stranded DNA template, can be synthesized.

[0016] In addition to an activity which adds dNTPs to DNA in the 5′to 3′direction (i.e., “polymerase” activity), many DNA polymerases alsopossess activities which remove dNTPs in the 5′to 3′ and/or the 3′ to5′direction (i.e., “exonuclease” activity). This dual activity ofcertain DNA polymerases is, however, a drawback for some in vitroapplications. For example, the in vitro synthesis of an intact copy of aDNA fragment by the polymerase activity, an elongation process whichproceeds in a 5′to 3′ direction along the template DNA strand, isjeopardized by the exonuclease activities which may simultaneously orsubsequently degrade the newly formed DNA.

[0017] Limitations of PCR-based Genotyping of Minisatellite,Microsatellite and STR DNA Sequences

[0018] Application of PCR-based methods to analysis of minisatellite orSTR DNA sequences has a number of significant limitations. It has beenshown, for example, that use of Taq and other thermostable DNApolymerases commonly employed in PCR and related automated amplificationmethods causes the accumulation of amplification products containingnon-templated 3′ terminal nucleotides (Clark, J. M., et al., J. Molec.Biol. 198:123-127 (1987); Clark, J. M., Nucl. Acids Res. 16:9677-9686(1988); Hu, G., DNA Cell Biol. 12:763-770 (1993)). That is, some of thenewly synthesized DNA strands produced in each round of amplificationhave had an extra nucleotide added to their 3′ termini, such that thenewly synthesized strands may be longer by one base.

[0019] Non-templated nucleotide addition is a slow process compared totemplate-directed synthesis (Clark, J. M., Nucl. Acids Res. 16:9677-9686(1988)), and its extent is sequence-dependent (Hu, G., DNA Cell Biol.12:763-770 (1993); Brownstein, M. J., et al., BioTechniques 20:1004-1010(1996)). Consequently, the PCR product is often heterogeneous in regardto extra nucleotide addition depending upon the primers and the reactionconditions used by the investigator (Magnuson, V. L., et al.,BioTechniques 21:700-709 (1996)). Extra nucleotide addition, incombination with “stutter” due to slippage during PCR amplification(Levinson, G., and Gutman, G. A., Molec. Biol. Evol. 4:203-221 (1987);Schlotterer, C., and Tautz, D., Nucl Acids Res. 20:211-215 (1992)),often results in complex DNA fragment patterns which are difficult tointerpret, especially by automated methods. This can result in impropergenotyping analysis, particularly if the percentage of non-templatednucleotide addition is between 30-70% of the PCR product (Smith, J. R.,et al., Genome Res. 5:312-317 (1995)).

[0020] Thus, a need currently exists for a rapid, automated method foridentifying, analyzing and typing polymorphic DNA fragments,particularly minisatellite, microsatellite or STR DNA fragments, thatwill not result in the problematic results described above. The presentinvention provides such a method.

BRIEF SUMMARY OF THE INVENTION

[0021] The present invention satisfies these needs in the art byproviding methods useful in the identification, analysis or typing ofpolymorphic DNA fragments, particularly minisatellite, microsatellite orSTR DNA fragments, in samples of DNA from a cell, particularly aeukaryotic cell. Specifically, the invention provides a method ofproducing a population of amplified DNA molecules, for use in analyzingor typing a DNA molecule in a DNA sample isolated from a cell,preferably a eukaryotic cell. The method of the present inventioncomprises contacting a DNA sample with a DNA polymerase (preferably athermostable DNA polymerases) reduced, substantially reduced oreliminated in the ability to add one or more non-templated nucleotidesto the 3′ terminus of a DNA molecule, amplifying a polymorphic DNAfragment, preferably a minisatellite, microsatellite or STR DNAfragment, within the DNA sample and analyzing the amplified polymorphicDNA fragment. In the method of the invention, the analysis step maycomprise, for example, sizing or sequencing the amplified DNA moleculeand optionally comparing the size and/or sequence of the amplified DNAmolecule to a different DNA sample which has been amplified according tothe invention. In preferred embodiments of the present invention, thethermostable DNA polymerase is a Thermotoga DNA polymerase, preferably aThermotoga DNA polymerase substantially reduced in 3′-5′ exonucleaseactivity, more preferably a Tne polymerase, a Tma polymerase, or amutant or derivative thereof, and most preferably a mutant of Tnepolymerase selected from the group consisting of Tne N′Δ219, D323A; TneN′Δ283, D323A; Tne N′Δ284, D323A; Tne N′Δ193, D323A; Tne D137A, D323A;Tne D8A, D323A; Tne G195D, D323A; Tne G37D, D323A; Tne N′Δ283; TneD137A, D323A, R722K; Tne D137A, D323A, R722Y; Tne D137A, D323A, R722L;Tne D137A, D323A, R722H; Tne D137A, D323A, R722Q; Tne D137A, D323A,F730Y; Tne D137A, D323A, K726R; Tne D137A, D323A, K726H; Tne D137A,D323A, R722K, F730Y; Tne D137A, D323A, R722K, K726R; Tne D137A, D323A,R722K, K726H; Tne D137A, D323A, R722H, F730Y; Tne D137A, D323A, R722H,K726R; Tne D137A, D323A, R722H, K726H; Tne D137A, D323A, R722Q, F730Y;Tne D137A, D323A, R722Q, K726R; Tne D137A, D323A, R722Q, K726H; TneD137A, D323A, R722N, F730Y; Tne D137A, D323A, R722N, K726R; Tne D137A,D323A, R722N, K726H; Tne D137A, D323A, F730S; Tne N′Δ283, D323A,R722K/H/Q/N/Y/L; Tne N′Δ219, D323A, R722K; Tne N′Δ219, D323A, F730Y; TneN′Δ219, D323A, K726R; Tne N′Δ219, D323A, K726H; Tne D137A, D323A, F730S,R722K/Y/Q/N/H/L, K726R/H; Tne D137A, D323A, F730T, R722K/Y/Q/N/H/L,K726R/H; Tne D137A, D323A, F730T; Tne F730S; Tne F730A, Tne K726R; TneK726H; and Tne D137A, D323A, R722N. The present invention isparticularly directed to the above methods wherein the eukaryotic cellis an plant cell or an animal cell, preferably a mammalian cell, morepreferably a normal, diseased, cancerous, fetal or embryonic mammaliancell, and most preferably a human cell. The invention is also directedto the above methods, further comprising isolating the polymorphic,minisatellite, microsatellite or STR DNA fragment and inserting it intoa vector, preferably an expression vector. By the present methods, thepolymorphic or microsatellite DNA fragment may be amplified prior tobeing inserted into the vector.

[0022] The present invention also provides a method of determining therelationship between a first individual and a second individual,comprising contacting a DNA sample from the first and second individualswith a DNA polymerase (e.g. a thermostable DNA polymerase) reduced,substantially reduced or eliminated in the ability to add one or morenon-templated nucleotides to the 3′terminus of a DNA molecule,amplifying one or more DNA molecules in the DNA sample to generate acollection of amplified polymorphic DNA fragments, separating theamplified DNA fragments by length, and comparing the pattern ofamplified DNA fragments from the first individual to that of the secondindividual. This method also allows the identification of one or moreunique polymorphic DNA fragments, particularly a minisatellite,microsatellite or STR DNA fragment, that is specifically present in onlyone of the two individuals. This method may further comprise determiningthe sequence of the unique polymorphic, minisatellite, microsatellite orSTR DNA fragment. In this embodiment of the present invention, thethermostable DNA polymerase may be a Thermotoga DNA polymerase,preferably a Thermotoga DNA polymerase substantially reduced in 3′-5′exonuclease activity, more preferably a Tne polymerase, a Tmapolymerase, or a mutant or derivative thereof, and most preferably amutant of Tne polymerase selected from the group consisting of TneN′Δ219, D323A; Tne N′Δ283, D323A; Tne N′Δ284, D323A; Tne N′Δ193, D323A;Tne D137A, D323A; Tne D8A, D323A; Tne G195D, D323A; Tne G37D, D323A; TneN′Δ283; Tne D137A, D323A, R722K; Tne D137A, D323A, R722Y; Tne D137A,D323A, R722L; Tne D137A, D323A, R722H; Tne D137A, D323A, R722Q; TneD137A, D323A, F730Y; Tne D137A, D323A, K726R; Tne D137A, D323A, K726H;Tne D137A, D323A, R722K, F730Y; Tne D137A, D323A, R722K, K726R; TneD137A, D323A, R722K, K726H; Tne D137A, D323A, R722H, F730Y; Tne D137A,D323A, R722H, K726R; Tne D137A, D323A, R722H, K726H; Tne D137A, D323A,R722Q, F730Y; Tne D137A, D323A, R722Q, K726R; Tne D137A, D323A, R722Q,K726H; Tne D137A, D323A, R722N, F730Y; Tne D137A, D323A, R722N, K726R;Tne D137A, D323A, R722N, K726H; Tne D137A, D323A, F730S; Tne N′Δ283,D323A, R722K/H/Q/N/Y/L; Tne N′Δ219, D323A, R722K; Tne N′Δ219, D323A,F730Y; Tne N′Δ219, D323A, K726R; Tne N′Δ219, D323A, K726H; Tne D137A,D323A, F730S, R722K/Y/Q/N/H/L, K726R/H; Tne D137A, D323A, F730T,R722K/Y/Q/N/H/L, K726R/H; Tne D137A, D323A, F730T; Tne F730S; Tne F730A;Tne K726R; Tne K726H; and Tne D137A, D323A, R722N. The present inventionis particularly directed to the above methods wherein the first orsecond individual is an animal or a plant, and most preferably whereinthe first or second individual is a human.

[0023] The present invention also provides isolated nucleic acidmolecules encoding mutant Tne DNA polymerase proteins, wherein themutant Tne DNA polymerase proteins have an amino acid sequence as setforth in any one of SEQ ID NOs: 4-10. The invention also provides mutantTne DNA polymerase proteins having an amino acid sequence as set forthin any one of SEQ ID NOs:4-10, most preferably a mutant Tne polymeraseprotein selected from the group consisting of Tne N′Δ283, D323A (SEQ IDNO:4); Tne N′Δ193, D323A (SEQ ID NO:5); Tne D137A, D323A (SEQ ID NO:6);Tne D8A, D323A (SEQ ID NO:7); Tne G195D, D323A (SEQ ID NO:8); Tne G37D,D323A (SEQ ID NO:9); and Tne N′Δ283 (SEQ ID NO:10). The invention alsorelates to nucleic acid molecules and the proteins encoded by suchnucleic acid molecules for mutant Tne polymerases selected from thegroup consisting of Tne n′Δ283; Tne D137A, D323A, R722K; Tne D137A,D323A, R722Y; Tne D137A, D323A, R722L; Tne D137A, D323A, R722H; TneD137A, D323A, R722Q; Tne D137A, D323A, F730Y; Tne D137A, D323A, K726R;Tne D137A, D323A, K726H; Tne D137A, D323A, R722K, F730Y; Tne D137A,D323A, R722K, K726R; Tne D137A, D323A, R722K, K726H; Tne D137A, D323A,R722H, F730Y; Tne D137A, D323A, R722H, K726R; Tne D137A, D323A, R722H,K726H; Tne D137A, D323A, R722Q, F730Y; Tne D137A, D323A, R722Q, K726R;Tne D137A, D323A, R722Q, K726H; Tne D137A, D323A, R722N, F730Y; TneD137A, D323A, R722N, K726R; Tne D137A, D323A, R722N, K726H; Tne D137A,D323A, F730S; Tne N′Δ283, D323A, R722K/H/Q/N/Y/L; Tne N′Δ219, D323A,R722K; Tne N′Δ219, D323A, F730Y; Tne N′Δ219, D323A, K726R; Tne N′Δ219,D323A, K726H; Tne D137A, D323A, F730S, R722K/Y/Q/N/H/L, K726R/H; TneD137A, D323A, F730T, R722K/Y/Q/N/H/L, K726R/H; Tne D137A, D323A, F730T;Tne F730S; Tne F730A; Tne K726R, Tne K726H; and Tne D137A, D323A, R722N.These mutations may be made to sequence ID NO:2 to produce the mutantpolymerases having the indicated amino acid mutations (where, forexample, “D137A” indicates that the Asp (D) residue at position 137 inSEQ ID NO:2 has been mutated to an Ala (A) residue, and, for example,“R722K/Y/Q/N/H/L” indicates that the Arg (R) residue at position 722 inSEQ ID NO:2 has been mutated to a Lys (K), Tyr (Y), Gin (Q), Asn (N),His (H) or Leu (L) residue).

[0024] The present invention also provides kits for the identification,analysis or typing of a polymorphic DNA fragment, particularly aminisatellite, microsatellite or STR DNA fragment, comprising a firstcontainer containing one or more DNA polymerases reduced, substantiallyreduced or eliminated in the ability to add non-templated 3′ terminalnucleotides. Kits according to the invention may contain additionalcontainers selected from the group consisting of a container containingone or more DNA primer molecules, a container containing one or moredeoxynucleoside triphosphates needed to synthesize a DNA moleculecomplementary to the DNA template, and a container containing a buffersuitable for identifying, analyzing or typing a polymorphic DNA fragmentby the methods of the invention. Any number of these components of thekit may be combined in a single or multiple containers to provide thekit of the invention. According to the invention, the DNA polymerase ofthe kit is preferably a Thermotoga DNA polymerase, more preferably aThermotoga DNA polymerase substantially reduced in 3′-5′ exonucleaseactivity, still more preferably a Tne polymerase, a Tma polymerase, or amutant or derivative thereof, and most preferably a mutant of Tnepolymerase selected from the group consisting of Tne N′Δ283; Tne D137A,D323A, R722K; Tne D137A, D323A, R722Y; Tne D137A, D323A, R722L; TneD137A, D323A, R722H; Tne D137A, D323A, R722Q; Tne D137A, D323A, F730Y;Tne D137A, D323A, K726R; Tne D137A, D323A, K726H; Tne D137A, D323A,R722K, F730Y; Tne D137A, D323A, R722K, K726R; Tne D137A, D323A, R722K,K726H; Tne D137A, D323A, R722H, F730Y; Tne D137A, D323A, R722H, K726R;Tne D137A, D323A, R722H, K726H; Tne D137A, D323A, R722Q, F730Y; TneD137A, D323A, R722Q, K726R; Tne D137A, D323A, R722Q, K726H; Tne D137A,D323A, R722N, F730Y; Tne D137A, D323A, R722N, K726R; Tne D137A, D323A,R722N, K726H; Tne D137A, D323A, F730S; Tne N′Δ283, D323A,R722K/H/Q/N/Y/L; Tne N′Δ219, D323A, R722K; Tne N′Δ219, D323A, F730Y; TneN′Δ219, D323A, K726R; Tne N′Δ219, D323A, K726H; Tne D137A, D323A, F730S,R722K/Y/Q/N/H/L, K726R/H; Tne D137A, D323A, F730T, R722K/Y/Q/N/H/L,K726R/H; Tne D137A, D323A, F730T; Tne F730S; Tne F730A; Tne K726R; TneK726H; and Tne D137A, D323A, R722N.

[0025] The present invention also relates generally to mutated ormodified polymerases (DNA or RNA polymerases) which have reduced,substantially reduced or eliminated ability to add one or morenon-templated nucleotides to the 3′ terminus of a synthesized nucleicacid molecule (compared to the corresponding wildtype, unmutated orunmodified polymerase). Preferably, such mutant or modified polymeraseshave substantially reduced ability to add one or more non-templatednucleotides to the 3′ terminus of a synthesized nucleic acid molecule.Such polymerases of the invention may be thermostable or mesophilicpolymerases. Thus, the present invention relates to such mutated ormodified polymerases and to kits containing such polymerases. Theinvention also relates to the use of such mutant or modified polymerasesin a number of procedures including DNA sequencing, amplificationreactions, nucleic acid synthesis, and polymorphism analysis.

[0026] Mutant or modified polymerases of particular interest in theinvention include Taq DNA polymerase, Tne DNA polymerase, Tma DNApolymerase, Pfu DNA polymerase, Tfl DNA polymerase, Tth DNA polymerase,Tbr DNA polymerase, Pwo DNA polymerase, Bst DNA polymerase, Bca DNApolymerase, VENT™ DNA polymerase, DEEP VENT™ DNA polymerase, T7 DNApolymerase, T5 DNA polymerase, DNA polymerase III, Klenow fragment DNApolymerase, Stoffel fragment DNA polymerase, and mutants, fragments orderivatives thereof RNA polymerases of interest include T7, SP6, and T3RNA polymerases and mutants, variants and derivatives thereof.

[0027] The present invention relates in particular to mutant PolI typeDNA polymerases (preferably thermostable DNA polymerases) wherein one ormore mutations have been made in the O-helix which reduces,substantially reduces or eliminates the ability of the enzyme to add oneor more non-templated nucleotides to the 3′ terminus of a synthesizednucleic acid molecule. The O-helix is defined as RXXXKXXXFXXXYX (SEQ IDNO:11), wherein X may be any amino acid. The preferred sites formutation or modification to produce the polymerases of the invention arethe R and/or F and/or K and/or Y positions in the O-helix, althoughother changes (or combinations thereof) within the O-helix may be madeto make the desired polymerase. In this preferred aspect of theinvention, R and/or F and/or K and/or Y may be replaced with any otheramino acid including Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

[0028] In accordance with the invention, other functional changes (orcombinations thereof) may be made to the polymerases having reducedability to add non-templated nucleotides to the 3′ terminus of asynthesized nucleic acid molecule. For example, the polymerase may alsobe modified to reduce, substantially reduce or eliminate 5′ exonucleaseactivity, and/or 3′ exonuclease activity. Thus, the invention relates tomutant or modified DNA polymerases having reduced ability to addnon-templated nucleotides which are modified in at least one wayselected from the group consisting of

[0029] (a) to reduce or eliminate the 3′-5′ exonuclease activity of thepolymerase;

[0030] (b) to reduce or eliminate the 5′-3′ exonuclease activity of thepolymerase;

[0031] Any one or a number of these mutations or modifications (orcombinations thereof) may be made to provide the polymerases of theinvention. Preferred polymerases of the invention, in addition to havingreduced ability to add non-templated 3′ nucleotides, also have reduced,substantially reduced or eliminated 3′ exonuclease activity.

[0032] The present invention is also directed to nucleic acid molecules(preferably vectors) containing a gene encoding the mutant or modifiedpolymerases of the present invention and to host cells containing suchmolecules. Any number of hosts may be used to express the gene ofinterest, including prokaryotic and eukaryotic cells. Preferably,prokaryotic cells are used to express the polymerases of the invention.The preferred prokaryotic host according to the present invention is E.coli.

[0033] The invention also relates to a method of producing thepolymerases of the invention, said method comprising:

[0034] (a) culturing the host cell comprising a gene encoding apolymerase of the invention;

[0035] (b) expressing said gene; and

[0036] (c) isolating said polymerase from said host cell.

[0037] The invention also relates to a method of synthesizing a nucleicacid molecule comprising:

[0038] (a) mixing one or more nucleic acid templates (e.g. RNA or DNA)with one or more polymerases of the invention; and

[0039] (b) incubating said mixture under conditions sufficient tosynthesize nucleic acid molecules complementary to all or a portion ofsaid templates. Such condition may include incubation with one or moredeoxy- and/or dideoxyribonucleoside triphosphates. Such deoxy- anddideoxyribonucleoside triphosphates include dATP, dCTP, dGTP, dTTP,dITP, 7-deaza-dGTP, 7-deaza-dATP, dUTP, ddATP, ddCTP, ddGTP, ddITP,ddTTP, [α-S]dATP, [α-S]dTTP, [α-S]dGTP, and [α-S]dCTP. The synthesizednucleic acid molecules may in accordance with the invention be clonedinto one or more vectors.

[0040] The invention also relates to a method of sequencing a DNAmolecule, comprising:

[0041] (a) hybridizing a primer to a first DNA molecule;

[0042] (b) contacting said molecule of step (a) with deoxyribonucleosidetriphosphates, one or more DNA polymerases of the invention, and one ormore terminator nucleotides;

[0043] (c) incubating the mixture of step (b) under conditionssufficient to synthesize a random population of DNA moleculescomplementary to said first DNA molecule, wherein said synthesized DNAmolecules are shorter in length than said first DNA molecule and whereinsaid synthesized DNA molecules comprise a terminator nucleotide at their3′ termini; and

[0044] (d) separating said synthesized DNA molecules by size so that atleast a part of the nucleotide sequence of said first DNA molecule canbe determined. Such terminator nucleotides include but are not limitedto dideoxyribonucleoside triphosphates such as ddTTP, ddATP, ddGTP,ddITP or ddCTP.

[0045] The invention also relates to a method for amplifying a doublestranded DNA molecule, comprising:

[0046] (a) providing a first and second primer, wherein said firstprimer is complementary to a sequence at or near the 3′-termini of thefirst strand of said DNA molecule and said second primer iscomplementary to a sequence at or near the 3′-termini of the secondstrand of said DNA molecule;

[0047] (b) hybridizing said first primer to said first strand and saidsecond primer to said second strand in the presence of one or morepolymerases of the invention, under conditions such that a third DNAmolecule complementary to said first strand and a fourth DNA moleculecomplementary to said second strand are synthesized;

[0048] (c) denaturing said first and third strands, and said second andfourth strands; and

[0049] (d) repeating steps (a) to (c) one or more times. The amplifieddouble-stranded nucleic acid molecules produced by the method of theinvention may be cloned into one or more vectors. Thus, the inventionrelates also to a method of cloning an amplified DNA moleculecomprising:

[0050] (a) amplifying one or more DNA molecules with one or morepolymerases of the invention; and

[0051] (b) ligating said amplified DNA molecules in one or more vectors.

[0052] The invention further relates to a method of cloning a nucleicacid molecule comprising:

[0053] (a) mixing a nucleic acid template (or one or more templates)with one or more polymerases of the invention;

[0054] (b) incubating said mixture under conditions sufficient tosynthesize a nucleic acid molecule complementary to all or a portion ofsaid template, thereby producing a double-stranded nucleic acid molecule(preferably a double-stranded DNA molecule); and

[0055] (c) ligating said double-stranded nucleic acid molecule into oneor more vectors.

[0056] Preferably, the vectors used for ligating the amplified orsynthesized double-stranded nucleic acid molecules have blunt endedtermini and may be prepared by digesting a vector with any one or anumber of restriction enzymes known in the art which provide blunt endcleavage. Such restriction enzymes include ScaI, SmaI, HpaI, HincII,HaeIII, AluI, and the like.

[0057] The invention also relates to kits for sequencing, amplifying,synthesizing or cloning of nucleic acid molecules comprising one or morepolymerases of the invention and one or more other components (orcombinations thereof) selected from the group consisting of

[0058] (a) one or more dideoxyribonucleoside triphosphates;

[0059] (b) one or more deoxyribonucleoside triphosphates;

[0060] (c) one or more primers;

[0061] (d) one or more suitable buffers; and

[0062] (e) one or more ligases.

[0063] Other preferred embodiments of the present invention will beapparent to one of ordinary skill in light of the following drawings anddescription of the invention, and of the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0064]FIG. 1 shows the restriction map of the approximate DNA fragmentwhich contains the Tne DNA polymerase gene in pSport 1 and pUC19. Thisfigure also shows the region containing the O-helix homologoussequences.

[0065]FIG. 2A schematically depicts the construction of plasmids pUC-Tne(3′→5′) and pUC-Tne FY.

[0066]FIG. 2B schematically depicts the construction of plasmidspTrcTne35 and pTrcTne FY.

[0067]FIG. 3 schematically depicts the construction of plasmid pTrcTne35FY.

[0068]FIG. 4 schematically depicts the construction of plasmidspTTQTne5FY and pTTQTne535FY.

[0069]FIG. 5 depicts a plasmid containing the Taq DNA polymerase gene.

[0070]FIG. 6 depicts an autoradiogram showing of the ability ofpolymerase mutants to add non-templated 3′ nucleotides.

[0071]FIG. 7 is an autoradiogram of the product of PCR amplification ofthe upper and lower alleles of the CD4 locus, using primerscorresponding to these alleles, demonstrating nontemplated nucleotideaddition (n+1) by Taq DNA polymerase but not by Tne DNA polymerase.

[0072]FIG. 8 is an autoradiogram of the product of PCR amplification ofthe upper and lower alleles of the D20S27 locus, using primerscorresponding to these alleles, demonstrating nontemplated nucleotideaddition (n+1) by Taq DNA polymerase but not by Tne DNA polymerase.

[0073]FIG. 9 is a composite of electropherogram gel scans of PCRamplifications at the D15S153 (FIGS. 9A and 9B) and D15S127 loci (FIGS.9C and 9D), demonstrating nontemplated nucleotide addition (n+1) by TaqDNA polymerase (FIGS. 9A and 9C) but not by Tne DNA polymerase (FIGS. 9Band 9D).

[0074]FIG. 10A and B are composites of a electropherogram gel scan ofPCR amplifications at D16S405 and D16S401 loci.

[0075]FIG. 11 is a composite of a electropherogram gel scan of PCRamplifications at D16S401 locus.

[0076]FIG. 12A and B are composites of a electropherogram gel scan ofPCR amplifications at D15S127 and D15S153 loci.

[0077]FIG. 13 is a composite of a electropherogram gel scan of PCRamplifications at D16S401 locus.

DETAILED DESCRIPTION OF THE INVENTION

[0078] Definitions

[0079] In the description that follows, a number of terms used inrecombinant DNA technology are extensively utilized. In order to providea clearer and consistent understanding of the specification and claims,including the scope to be given such terms, the following definitionsare provided.

[0080] Polymorphic. As is understood by one of ordinary skill in theart, a nucleic acid molecule is said to be “polymorphic” if it may existin more than one form. For example, a nucleic acid molecule is said tobe polymorphic if it may have more than one specific nucleotide sequence(such as degenerate nucleic acid molecules or genes that may each encodethe same protein). More commonly, a nucleic acid molecule is said to bepolymorphic if it displays size differences (i.e., differences inlength), particularly when comparisons of nucleic acid molecules fromdifferent individuals are made. Of course, other definitions of the term“polymorphic” will be apparent to one of ordinary skill and are alsoencompassed within this definition.

[0081] Cloning vector. A plasmid, cosmid or phage DNA or other DNAmolecule which is able to replicate autonomously in a host cell, andwhich is characterized by one or a small number of restrictionendonuclease recognition sites at which such DNA sequences may be cut ina determinable fashion without loss of an essential biological functionof the vector, and into which DNA may be spliced in order to bring aboutits replication and cloning. The cloning vector may further contain amarker suitable for use in the identification of cells transformed withthe cloning vector. Markers, for example, are tetracycline resistance orampicillin resistance.

[0082] Recombinant host. Any prokaryotic or eukaryotic microorganismwhich contains the desired cloned genes in an expression vector, cloningvector or any DNA molecule. The term “recombinant host” is also meant toinclude those host cells which have been genetically engineered tocontain the desired gene on the host chromosome or genome.

[0083] Host. Any prokaryotic or eukaryotic microorganism that is therecipient of a replicable expression vector, cloning vector or any DNAmolecule. The DNA molecule may contain, but is not limited to, astructural gene, a promoter and/or an origin of replication.

[0084] Promoter. A DNA sequence generally described as the 5′ region ofa gene, located proximal to the start codon. At the promoter region,transcription of an adjacent gene(s) is initiated.

[0085] Gene. A DNA sequence that contains information necessary forexpression of a polypeptide or protein. It includes the promoter and thestructural gene as well as other sequences involved in expression of theprotein.

[0086] Structural gene. A DNA sequence that is transcribed intomessenger RNA that is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

[0087] Operably linked. As used herein “operably linked” means that thepromoter is positioned to control the initiation of expression of thepolypeptide encoded by the structural gene.

[0088] Expression. Expression is the process by which a gene produces apolypeptide. It includes transcription of the gene into messenger RNA(mRNA) and the translation of such mRNA into polypeptide(s).

[0089] Substantially Pure. As used herein “substantially pure” meansthat the desired purified protein is essentially free from contaminatingcellular contaminants which are associated with the desired protein innature. Contaminating cellular components may include, but are notlimited to, phosphatases, exonucleases, endonucleases or undesirable DNApolymerase enzymes.

[0090] Primer. As used herein “primer” refers to a single-strandedoligonucleotide that is extended by covalent bonding of nucleotidemonomers during amplification or polymerization of a nucleic acidmolecule. Minisatellite primers used for the amplification ofminisatellite dimer, trimer, tetramer, etc., sequences are well-known inthe art.

[0091] Template. The term “template” as used herein refers to adouble-stranded or single-stranded nucleic acid molecule which is to beamplified, synthesized or sequenced. In the case of a double-strandedDNA molecule, denaturation of its strands to form a first and a secondstrand is performed before these molecules may be amplified, synthesizedor sequenced. A primer, complementary to a portion of a template ishybridized under appropriate conditions and the polymerase of theinvention may then synthesize a molecule complementary to said templateor a portion thereof The newly synthesized molecule, according to theinvention, may be equal or shorter in length than the original template.Mismatch incorporation or strand slippage during the synthesis orextension of the newly synthesized molecule may result in one or anumber of mismatched base pairs. Thus, the synthesized molecule need notbe exactly complementary to the template.

[0092] Incorporating. The term “incorporating” as used herein meansbecoming a part of a nucleic acid (e.g., DNA) molecule or primer.

[0093] Amplification. As used herein “amplification” refers to any invitro method for increasing the number of copies of a nucleotidesequence with the use of a DNA polymerase. Nucleic acid amplificationresults in the incorporation of nucleotides into a DNA molecule orprimer thereby forming a new DNA molecule complementary to a DNAtemplate. The formed DNA molecule and its template can be used astemplates to synthesize additional DNA molecules. As used herein, oneamplification reaction may consist of many rounds of DNA replication.DNA amplification reactions include, for example, polymerase chainreactions (PCR). One PCR reaction may consist of 5 to 100 “cycles” ofdenaturation and synthesis of a DNA molecule.

[0094] Oligonucleotide. “Oligonucleotide” refers to a synthetic ornatural molecule comprising a covalently linked sequence of nucleotideswhich are joined by a phosphodiester bond between the 3′ position of thepentose of one nucleotide and the 5′ position of the pentose of theadjacent nucleotide.

[0095] Nucleotide. As used herein “nucleotide” refers to abase-sugar-phosphate combination. Nucleotides are monomeric units of anucleic acid sequence (DNA and RNA). The term nucleotide includesdeoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP,dTTP, or derivatives thereof. Such derivatives include, for example,[αS]dATP, 7-deaza-dGTP and 7-deaza-dATP. The term nucleotide as usedherein also refers to dideoxyribonucleoside triphosphates (ddNTPs) andtheir derivatives. Illustrated examples of dideoxyribonucleosidetriphosphates include, but are not limited to, ddATP, ddCTP, ddGTP,ddITP, and ddTTP. According to the present invention, a “nucleotide” maybe unlabeled or detectably labeled by well known techniques. Detectablelabels include, for example, radioactive isotopes, fluorescent labels,chemiluminescent labels, bioluminescent labels and enzyme labels.

[0096] Thermostable. As used herein “thermostable” refers to apolymerase which is resistant to inactivation by heat. DNA polymerasessynthesize the formation of a DNA molecule complementary to asingle-stranded DNA template by extending a primer in the 5′-to-3′direction. This activity for mesophilic DNA polymerases may beinactivated by heat treatment. For example, T5 DNA polymerase activityis totally inactivated by exposing the enzyme to a temperature of 90° C.for 30 seconds. As used herein, a thermostable polymerase activity ismore resistant to heat inactivation than a mesophilic polymerase.However, a thermostable polymerase does not mean to refer to an enzymewhich is totally resistant to heat inactivation and thus heat treatmentmay reduce the polymerase activity to some extent. A thermostablepolymerase typically will also have a higher optimum temperature thanmesophilic polymerases.

[0097] Hybridization. The terms “hybridization” and “hybridizing” refersto the pairing of two complementary single-stranded nucleic acidmolecules (RNA and/or DNA) to give a double-stranded molecule. As usedherein, two nucleic acid molecules may be hybridized, although the basepairing is not completely complementary. Accordingly, mismatched basesdo not prevent hybridization of two nucleic acid molecules provided thatappropriate conditions, well known in the art, are used. In the presentinvention, the term “hybridization” refers particularly to hybridizationof an oligonucleotide to a template molecule.

[0098] 3′-5′ Exonuclease Activity. “3′-5′ exonuclease activity” is anenzymatic activity well known to the art. This activity is oftenassociated with DNA polymerases, and is thought to be involved in a DNAreplication “editing” or correction mechanism.

[0099] A “DNA polymerase substantially reduced in 3′-5′ exonucleaseactivity” (which may also be represented as “3′exo−”) is defined hereinas either (1) a mutated DNA polymerase that has about or less than 10%,or preferably about or less than 1%, of the 3′-5′ exonuclease activityof the corresponding unmutated, wildtype enzyme, or (2) a DNA polymerasehaving a 3′-5′ exonuclease specific activity which is less than about 1unit/mg protein, or preferably about or less than 0.1 units/mg protein.A unit of activity of 3′-5′ exonuclease is defined as the amount ofactivity that solubilizes 10 nmoles of substrate ends in 60 min. at 37°C., assayed as described in the “BRL 1989 Catalogue & Reference Guide”,page 5, with HhaI fragments of lambda DNA 3′-end labeled with [³H]dTTPby terminal deoxynucleotidyl transferase (TdT). Protein is measured bythe method of Bradford, Anal. Biochem. 72:248 (1976). As a means ofcomparison, natural, wildtype T5-DNA polymerase (DNAP) or T5-DNAPencoded by pTTQ19-T5-2 has a specific activity of about 10 units/mgprotein while the DNA polymerase encoded by pTTQ19-T5-2(exo⁻) (U.S. Pat.No. 5,270,179) has a specific activity of about 0.0001 units/mg protein,or 0.001% of the specific activity of the unmodified enzyme, a 10⁵-foldreduction.

[0100] 5′-3′ Exonuclease Activity. “5′-3′ exonuclease activity” is alsoan enzymatic activity well known in the art. This activity is oftenassociated with DNA polymerases, such as E. coli PolI and PolIII.

[0101] A “DNA polymerase substantially reduced in 5′-3′exonucleaseactivity” (which may also be represented as “5′exo−”) is defined hereinas either (1) a mutated DNA polymerase that has about or less than 10%,or preferably about or less than 1%, of the 5′-3′exonuclease activity ofthe corresponding unmutated, wildtype enzyme, or (2) a DNA polymerasehaving 5′-3′ exonuclease specific activity which is less than about 1unit/mg protein, or preferably about or less than 0.1 units/mg protein.

[0102] Both of the 3′-5′ and 5′-3′ exonuclease activities can beobserved on sequencing gels. Active 5′-3′ exonuclease activity willproduce nonspecific ladders in a sequencing gel by removing nucleotidesfrom the 5′-end of the growing primers. 3′-5′ exonuclease activity canbe measured by following the degradation of radiolabeled primers in asequencing gel. Thus, the relative amounts of these activities, e.g. bycomparing wildtype and mutant polymerases, can be determined with nomore than routine experimentation.

[0103] Minisatellite DNA. As used herein, the term “minisatellite DNA”refers to a DNA fragment comprising a short stretch of tandemlyrepetitive nucleotide sequence. In vivo, minisatellite DNA fragments arefound interspersed throughout the genomes of most eukaryotic organismsthus far examined. These repeating sequences appear in tandem and oftenin variable numbers within the genome; thus, the terms “short tandemrepeats” (“STRs”) or “variable numbers of tandem repeats” (“VNTRs”) maybe used synonymously when referring to these regions. Minisatellite DNAfragments are typically about 9 bases to about 60 bases in length andare repeated about 20-50 times at a typical locus in a eukaryoticgenome.

[0104] Microsatellite DNA. As used herein, the term “microsatellite DNA”refers to DNA fragments which are typically of a repeat unit size ofabout 1-6 bases in length. The most prevalent of these microsatelliteDNA fragments in the human genome is the dinucleotide repeat(dC-dA)_(n)•(dG-dT)_(n) (where n is the number of repetitions in a givenstretch of nucleotides). The terms “STRs” and “VNTRs” may also be usedsynonymously to denote these structures.

[0105] Non-templated 3′ Terminal Nucleotide Addition. As used herein,the term “non-templated 3′ terminal nucleotide addition” or“extranucleotide addition” means the propensity of an enzyme such as aDNA polymerase to incorporate one or more additional nucleotides, whichare not found in the template strand at the 3′ terminus of a newlysynthesized nucleic acid molecule in a synthesis or amplificationreaction, such as PCR. As a result of non-templated 3′ terminalnucleotide addition, the synthesized or amplification products (i.e.,the newly synthesized DNA strand) will be longer by one or morenucleotides than is the template, in such a fashion that if the templateis “n” nucleotides in length, the synthesis or amplification productswill be “n+1,” “n+2,” “n+3,” etc., nucleotides in length. A “polymerasesubstantially reduced in the ability to add one or more non-templatednucleotides to the 3′ terminus of a nucleic acid molecule” is definedherein as a DNA polymerase, which when it has no 3′ exonuclease activityor has substantially reduced 3′ exonuclease activity, it will produce acollection of amplification products in which less than about 50%,preferably less than about 30%, more preferably less than about 20%,still more preferably less than about 10%, still more preferably lessthan about 5%, and most preferably less than about 1% of theamplification products contain one or more non-templated nucleotides attheir 3′ termini compared to amplification products produced by Taq DNApolymerase assayed under the same conditions. Preferably, the conditionsused for assaying 3′ non-templated nucleotide addition is performed suchthat less than 100% of the amplification products of Taq DNA polymeraseexhibits 3′ non-templated nucleotide addition. Included in thisdefinition are those polymerases that satisfy this definition for anyprimer set used. Thus, if the use of any primer set provides theindicated reduction of 3′ non-templated nucleotide addition, thepolymerase is said to be substantially reduced in the ability to add oneor more non-templated nucleotides to the 3′ terminus of a nucleic acidmolecule.

[0106] When referring to polymerases which have been mutated or modifiedto reduce or eliminate 3′ non-templated nucleotide addition, the mutatedor modified polymerase is said to be “reduced in the ability to add oneor more non-templated nucleotides to the 3′ terminus of a nucleic acidmolecule” when the polymerase has a lower or reduced or eliminatedability to add non-templated 3′ nucleotides compared to thecorresponding unmutated, unmodified or wildtype polymerase. For example,when testing the affect of a point mutation in the O-helix of apolymerase on non-templated nucleotide addition, the polymeraseunmodified in the same position of the O-helix is preferably used forcomparison purposes. Such mutated or modified polymerases are said to“substantially reduced in the ability to add one or more non-templatednucleotides to the 3′ terminus of a nucleic acid molecule” if themutated or modified polymerase has less than about 50%, preferably lessthan about 30%, more preferably less than about 20%, still morepreferably less than about 10%, still more preferably less than about5%, and most preferably less than about 1% of the activity for addingnon-templated 3′ terminal nucleotides compared to the correspondingunmutated, unmodified or wildtype polymerase. Preferably, the conditionsused for assaying 3′ non-templated nucleotide addition is performed suchthat less than 100% of the amplification products produced by theunmutated, unmodified or wildtype polymerase control exhibits 3′non-templated nucleotide addition. Included in this definition are thosemutant or modified polymerases that satisfy this definition for anyprimer set tested.

[0107] The ability of a polymerase to add a non-templated 3′ terminalnucleotide to the growing strand may be assessed by a variety oftechniques, most preferably by gel electrophoresis of the synthesized oramplification products for a direct size comparison and by comparison tomarkers of known size (see FIGS. 6-13).

[0108] Other terms used in the fields of recombinant DNA technology andmolecular and cell biology as used herein will be generally understoodby one of ordinary skill in the applicable arts.

[0109] Sources of Polymerases

[0110] The methods of the present invention rely on the use ofpolymerases (thermostable or mesophilic DNA or RNA polymerases) reduced,substantially reduced or eliminated in the ability to add one or morenon-templated 3′ terminal nucleotide to a growing nucleic acid strand.These thermostable DNA polymerases may be obtained from any strain ofany thermophilic microorganism, including but not limited to strains ofThermus aquaticus (Taq polymerase; see U.S. Pat. Nos. 4,889,818 and4,965,188), Thermus thermophilus (Tth polymerase), Thernococcuslitoralis (Tli or VENT™ polymerase), Pyrococcus furiosus (Pfu orDEEPVENT™ polymerase), Pyrococcus woosii (Pwo polymerase) and otherPyrococcus species, Bacillus sterothermophilus (Bst polymerase),Sulfolobus acidocaldarius (Sac polymerase), Thermoplasma acidophilum(Tac polymerase), Bacillus caldophilus (Bca polymerase), Thermus flavus(Tfl/Tub polymerase), Thermus ruber (Tru polymerase), Thermus brockianus(DYNAZYME™ polymerase), Thermotoga neapolitana (Tne polymerase; see WO96/10640 and WO96/41014), Thermotoga maritima (Tma polymerase; see U.S.Pat. No. 5,374,553) and other species of the Thermotoga genus (Tsppolymerase) and Methanobacterium thermoautotrophicum (Mth polymerase).Mesophilic DNA polymerases of interest in the invention include but arenot limited to T7 DNA polymerases, T5 DNA polymerase, DNA polymeraseIII, Klenow fragment DNA polymerase and mutants, fragments orderivatives thereof RNA polymerases such as T3, T5, SP6 and mutants,variants and derivatives thereof may also be used in accordance with theinvention. Polymerases having reduced or substantially reduced abilityto add a non-templated 3′ nucleotide to a growing nucleic acid strandmay be wildtype polymerases, or may be made by mutating such wildtypepolymerases by standard techniques (for example, by generating pointmutations, insertions, deletions, etc., in the wildtype gene orprotein). Polymerases that are reduced or substantially reduced in theability to add a non-templated 3′ nucleotide to a growing strand may beidentified by assaying the synthesized products (e.g. PCR products)formed by such enzymes, as is well-known in the art and as generallydescribed below in the Examples.

[0111] The nucleic acid polymerases used in the present invention may bemesophilic or thermophilic, and are preferably thermophilic. Preferredmesophilic DNA polymerases include T7 DNA polymerase, T5 DNA polymerase,Klenow fragment DNA polymerase, DNA polymerase III and the like.Preferred thermostable DNA polymerases that may be used in the methodsof the invention include Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragment,VENT™ and DEEPVENT™ DNA polymerases, and mutants, variants andderivatives thereof (U.S. Pat. Nos. 5,436,149; 4,889,818; 4,965,188;5,079,352; 5,614,365; 5,374,553; 5,270,179; 5,047,342; 5,512,462; WO92/06188; WO 92/06200; WO 96/10640; Barnes, W. M., Gene 112:29-35(1992); Lawyer, F. C., et al., PCR Meth. Appl. 2:275-287 (1993); Flaman,J.-M, et al., Nucl. Acids Res. 22(15):3259-3260 (1994)). Foramplification of long nucleic acid molecules (e.g., nucleic acidmolecules longer than about 3-5 Kb in length), at least two DNApolymerases (one substantially lacking 3′ exonuclease activity and theother having 3′ exonuclease activity) are typically used. See U.S. Pat.Nos. 5,436,149; 5,512,462; Farnes, W. M., Gene 112:29-35 (1992); andcopending U.S. patent application Ser. No. 08/689,814, filed Feb. 14,1997, the disclosures of which are incorporated herein in theirentireties. Examples of DNA polymerases substantially lacking in 3′exonuclease activity include, but are not limited to, Taq, Tne(exo⁻),Tma(exo⁻), Pfu (exo⁻), Pwo(exo⁻) and Tth DNA polymerases, and mutants,variants and derivatives thereof.

[0112] Polypeptides having nucleic acid polymerase activity arepreferably used in the present methods at a final concentration insolution of about 0.1-200 units per milliliter, about 0.1-50 units permilliliter, about 0.1-40 units per milliliter, about 0.1-3.6 units permilliliter, about 0.1-34 units per milliliter, about 0.1-32 units permilliliter, about 0.1-30 units per milliliter, or about 0.1-20 units permilliliter, and most preferably at a concentration of about 20-40 unitsper milliliter. Of course, other suitable concentrations of nucleic acidpolymerases suitable for use in the invention will be apparent to one orordinary skill in the art.

[0113] In a preferred aspect of the invention, polymerases of theinvention and preferably the mutant or modified polymerases of theinvention are made by recombinant techniques. A number of clonedpolymerase genes are available or may be obtained using standardrecombinant techniques.

[0114] To clone a gene encoding a polymerase, which may be modified inaccordance with the invention, isolated DNA which contains thepolymerase gene is used to construct a recombinant library in a vector.Any vector, well known in the art, can be used to clone the DNApolymerase of interest. However, the vector used must be compatible withthe host in which the recombinant DNA library will be transformed.

[0115] Prokaryotic vectors for constructing the plasmid library includeplasmids such as those capable of replication in E. coli such as, forexample, pBR322, ColE1, pSC101, pUC-vectors (pUC18, pUC19, etc.: In:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1982); and Sambrook et al., In:Molecular Cloning A Laboratory Manual (2d ed.) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). Bacillus plasmidsinclude pC194, pC221, pC217, etc. Such plasmids are disclosed byGlyczan, T. In: The Molecular Biology Bacilli, Academic Press, York(1982), 307-329. Suitable Streptomyces plasmids include pIJ101 (Kendallet al., J. Bacteriol 169:4177-4183 (1987)). Pseudomonas plasmids arereviewed by John et al., (Rad. Insec. Dis. 8:693-704 (1986)), and Igaki,(Jpn. J. Bacteriol. 33:729-742 (1978)). Broad-host range plasmids orcosmids, such as pCP13 (Darzins and Chakrabarbary, J. Bacteriol.159:9-18, 1984) can also be used for the present invention. Thepreferred vectors for cloning the genes of the present invention areprokaryotic vectors. Preferably, pCP13 and pUC vectors are used to clonethe genes of the present invention.

[0116] The preferred host for cloning the polymerase genes of interestis a prokaryotic host. The most preferred prokaryotic host is E. coli.However, the desired polymerase genes of the present invention may becloned in other prokaryotic hosts including, but not limited to,Escherichia, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia,and Proteus. Bacterial hosts of particular interest include E. coliDH10B, which may be obtained from Life Technologies, Inc. (LTI)(Rockville, Md).

[0117] Eukaryotic hosts for cloning and expression of the polymerases ofinterest include yeast, fungi, and mammalian cells. Expression of thedesired polymerase in such eukaryotic cells may require the use ofeukaryotic regulatory regions which include eukaryotic promoters.Cloning and expressing the polymerase gene in eukaryotic cells may beaccomplished by well known techniques using well known eukaryotic vectorsystems.

[0118] Once a DNA library has been constructed in a particular vector,an appropriate host is transformed by well known techniques. Transformedcolonies are preferably plated at a density of approximately 200-300colonies per petri dish. For thermostable polymerase selection, coloniesare then screened for the expression of a heat stable DNA polymerase bytransferring transformed E. coli colonies to nitrocellulose membranes.After the transferred cells are grown on nitrocellulose (approximately12 hours), the cells are lysed by standard techniques, and the membranesare then treated at 95° C. for 5 minutes to inactivate the endogenous E.coli enzyme. Other temperatures may be used to inactivate the hostpolymerases depending on the host used and the temperature stability ofthe polymerase to be cloned. Stable polymerase activity is then detectedby assaying for the presence of polymerase activity using well knowntechniques (see, e.g., Sagner et al., Gene 97:119-123 (1991), which ishereby incorporated by reference in its entirety). The gene encoding apolymerase of the present invention can be cloned using the proceduredescribed by Sanger et al., supra. Other techniques for selecting clonedpolymerases in accordance with the present invention will be well-knownto those of ordinary skill in the art.

[0119] Modifications or Mutations of Polymerases

[0120] In accordance with the invention, the nucleotide binding domainof the polymerase of interest is modified or mutated in such a way as toproduce a mutated or modified polymerase having reduced, substantiallyreduced or eliminated activity for adding non-templated 3′ nucleotides.The O-helix region typically defines the nucleotide binding domain ofDNA polymerases. The O-helix maybe defined as RXXXKXXXFXXXYX (SEQ IDNO:11), wherein X may be any amino acid. One or more mutations orcombinations of mutations may be made in the O-helix of any polymerasein order to reduce or eliminate on-templated 3′ nucleotide addition inaccordance with the invention. Such mutations include point mutation,frame-shift mutations, deletions and insertions. Preferably, one or morepoint mutations, resulting in one or more amino acid substitutions, areused to produce polymerases having such activity. Such mutations may bemade by a number of methods that will be familiar to one of ordinaryskill, including but not limited to site-directed mutagenesis. In apreferred aspect of the invention, one or more mutations at positions R,K, F, and/or Y in the polymerase O-helix may be made to produced apolymerase having the desired activity. Most preferably, one or moremutations at position R and/or F and/or K and/or Y within the O-helixresults in polymerases having reduced, substantially reduced oreliminated activity for adding non-templated 3′ nucleotides. In thepreferred aspect, amino acid substitutions are made at position R and/orF and/or K and/or Y (or combinations thereof). Thus, R (Arg) and/or F(Phe) and/or K (Lys) may be substituted with any other amino acidincluding Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. Preferably, R (Arg) issubstituted with amino acids Lys, Tyr, Leu, His, Gln, Met, or Asn. F(Phe) is preferably substituted with amino acids Tyr, Ala, Leu, Thr, andSer. K (Lys) is preferably substituted with amino acids Arg, Tyr, Leu,His, Gln, Met or Asn, and more preferably with Arg or His. Y (Tyr) ispreferably substituted with amino acids Lys, Arg, Ala, Thr, Phe, Leu,His, Gln, Met, or Asn. Positions corresponding to R, K, F and Y for RNApolymerases may also be determined by comparing nucleotide and/or aminoacid sequences with those of DNA polymerases, to determine homologiestherebetween. Corresponding mutations or modification may then be madeto produce the desired result in any RNA polymerase.

[0121] The O-helix has been identified and defined for a number ofpolymerases and may be readily identified for other polymerases by onewith skill in the art. Thus, given the defined O-helix region and themethods and assays described herein, one with skill in the art can makeone or a number of modifications which would result in polymeraseshaving reduced, substantially reduced or eliminated activity for addingnon-templated 3′ nucleotides. Accordingly, the invention relates tomethods for producing such polymerases having modifications in theO-helix domain resulting in reduction, substantial reduction orelimination of activity for adding non-templated 3′ nucleotides, methodsfor producing nucleic acid molecules encoding such polymerases, andpolymerases and nucleic acid molecules produced by such methods.

[0122] The following table illustrates identified O-helix regions forknown polymerases. Polymerase O-Helix Region SEQ ID NO. Po1I 754RRSAKAINFGLIYG 12 Taq 659 RRAAKTINFGVLYG 13 T7 518 RDNAKTFIYGFLYG 14 Tne722 RRVGKMVNFSIIYG 15 T5 588 RQAAKAITFGILYG 16 Tma 722 RRAGKMVNFSIIYG 17

[0123] Thus, in accordance with a preferred aspect of the invention,corresponding mutations in the R and/or F and/or K positions of theO-helix can be made for the following enzymes based on the tables below.Polymerase Mutation Position PolI Arg⁷⁵⁴ T5 Arg⁵⁸⁸ T7 Arg⁵¹⁸ Taq Arg⁶⁵⁹Tne Arg⁷²² Tma Arg⁷²² Bca Arg⁷⁰⁵ Bst Arg⁷⁰² Tth Arg⁶⁶¹ PolI Phe⁷⁶² T5Phe⁵⁹⁶ T7 Phe⁵²⁸ Taq Phe⁶⁶⁷ Tne Phe⁷³⁰ Tma Phe⁷³⁰ Bca Phe⁷¹³ Bst Phe⁷¹⁰Tth Phe⁶⁶⁹ PolI Lys⁷⁵⁸ T5 Lys⁵⁹² T7 Lys⁵²² Taq Lys⁶⁶³ Tne Lys⁷²⁶ TmaLys⁷²⁶ Bca Lys⁷⁰⁷ Bst Lys⁷⁰⁶ Tth Lys⁶⁶⁵

[0124] The mutation position of Arg⁷⁰⁵ for Bca is based on the sequenceinformation in GenBank. It should be noted, however, that according tothe sequence described by Vemori et al. J. Biochem. (Japan)113:401-410(1993), the position of Arg in Bca is 703.

[0125] Additional Modifications or Mutations of Polymerases

[0126] In accordance with the invention, in addition to the mutations ormodifications described above, one or more additional mutations ormodifications (or combinations thereof) may be made to the polymerasesof interest. Mutations or modifications of particular interest includethose modifications of mutations which (1) reduce or eliminate 3′to 5′exonuclease activity; and (2) reduce or eliminate 5′to 3′ exonucleaseactivity.

[0127] If the DNA polymerase has 3′-to-5′ exonuclease activity, thisactivity may be reduced, substantially reduced, or eliminated bymutating the polymerase gene. Such mutations include point mutations,frame shift mutations, deletions and insertions. Preferably, the regionof the gene encoding the 3′-to-5′ exonuclease activity is mutated ordeleted using techniques well known in the art (Sambrook et al., (1989)in: Molecular Cloning, A Laboratory Manual (2nd Ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

[0128] The 3′-to-5′ exonuclease activity can be reduced or impaired bycreating site specific mutants within the 3′→5′ exonuclease domain. Seeinfra. In a specific embodiment of the invention Asp³²³ of Tne DNApolymerase is changed to any amino acid, preferably to Ala³²³ tosubstantially reduce 3′→5′ exonuclease activity. In another specificembodiment of the invention, Asp³²³ of Tma may be changed to any otheramino acid, preferably to Ala to substantially reduce 3′→5′ exonucleaseactivity. The following represents a domain of interest for a number ofpolymerases for preparing 3′→5′ exonuclease mutants. Tne 318 PSFALDLETSS328 (SEQ ID NO:18) Po1 I 350 PVFAFDTETDS 360 (SEQ ID NO:19) T5 159GPVAFDSETSA 169 (SEQ ID NO:20) T7 1 MIVSDIEANA 10 (SEQ ID NO:21)

[0129] Mutations, such as insertions, deletions and substitutions withinthe above domain can result in substantially reduced 3′→5′ exonucleaseactivity. By way of example, Asp³⁵⁵ (PolI), Asp¹⁶⁴ (T5), and Asp⁵ (T7)may be substituted with any amino acid to substantially reduce 3′→5′exonuclease activity. For example, Asp at these positions may besubstituted with Ala.

[0130] The 5′→3′ exonuclease activity of the polymerases can be reduced,substantially reduced or eliminated by mutating the polymerase gene orby deleting the 5′to 3′exonuclease domain. Such mutations include pointmutations, frame shift mutations, deletions, and insertions. Preferably,the region of the gene encoding the 5′→3′ exonuclease activity isdeleted using techniques well known in the art. In embodiments of thisinvention, any one of six conserved amino acids that are associated withthe 5′→3′ exonuclease activity can be mutated. Examples of theseconserved amino acids with respect to Tne DNA polymerase include Asp⁸,Glu¹¹², Asp¹¹⁴, Asp¹¹⁵, Asp¹³⁷, and Asp¹³⁹. Other possible sites formutation are Gly¹⁰², Gly¹⁸⁷ and Gly¹⁹⁵.

[0131] Corresponding amino acid to target for other polymerases toreduce or eliminate 5′→3′ exonuclease activity as follows: E. coli po1I:Asp¹³, Glu¹¹³, Asp¹¹⁵, Asp¹¹⁶, Asp^(138,) and Asp¹⁴⁰. Taq po1: Asp¹⁸,Glu^(117,) Asp¹¹⁹, Asp¹²⁰, Asp¹⁴², and Asp¹⁴⁴. Tma po1: Asp⁸, Glu¹¹²,Asp¹¹⁴, Asp¹¹⁵, Asp¹³⁷, and Asp¹³⁹

[0132] Amino acid residues of Taq DNA polymerase are as numbered in U.S.Pat. No. 5,079,352. Amino acid residues of Thermotogamaritima (Tma) DNApolymerase are numbered as in U.S. Pat. No. 5,374,553.

[0133] Examples of other amino acids which may be targeted for otherpolymerases to reduce 5′→3′ exonuclease activity include: Enzyme orsource Mutation positions Streptococcus pneumoniae Asp¹⁰, Glu¹¹⁴,Asp¹¹⁶, Asp¹¹⁷, Asp¹³⁹, Asp¹⁴¹ Thermus flavus Asp¹⁷, Glu¹¹⁶, Asp¹¹⁸,Asp¹¹⁹, Asp¹⁴¹, Asp¹⁴³ Thermus thermophilus Asp¹⁸, Glu¹¹⁸, Asp¹²⁰,Asp¹²¹, Asp¹⁴³, Asp¹⁴⁵ Deinococcus radiodurans Asp¹⁸, Glu¹¹⁷, Asp¹¹⁹,Asp¹²⁰, Asp¹⁴², Asp¹⁴⁴ Bacillus caldotenax Asp⁹, Glu¹⁰⁹, Asp¹¹¹, Asp¹¹²,Asp¹³⁴, Asp¹³⁶

[0134] Coordinates of S. pneumoniae, T. flavus, D. radiodurans, B.caldotenax were obtained from Gutman and Minton. Coordinates of T.thermophilus were obtained from International Patent No. WO 92/06200.

[0135] Typically, the mutant polymerases of the invention can beaffected by substitution of amino acids typically which have differentproperties. For example, an acidic amino acid such as Asp may be changedto a basic, neutral or polar but uncharged amino acid such as Lys, Arg,His (basic); Ala, Val, Leu, Ile, Pro, Met, Phe, Trp (neutral); or Gly,Ser, Thr, Cys, Tyr, Asn or Gln (polar but uncharged). Glu may be changedto Asp, Ala, Val Leu, Ile, Pro, Met, Phe, Trp, Gly, Ser, Thr, Cys, Tyr,Asn or Gln.

[0136] Preferably, oligonucleotide directed mutagenesis is used tocreate the mutant polymerases which allows for all possible classes ofbase pair changes at any determined site along the encoding DNAmolecule. In general, this technique involves annealing aoligonucleotide complementary (except for one or more mismatches) to asingle stranded nucleotide sequence coding for the DNA polymerase ofinterest. The mismatched oligonucleotide is then extended by DNApolymerase, generating a double stranded DNA molecule which contains thedesired change in the sequence on one strand. The changes in sequencecan of course result in the deletion, substitution, or insertion of anamino acid. The double stranded polynucleotide can then be inserted intoan appropriate expression vector, and a mutant polypeptide can thus beproduced. The above-described oligonucleotide directed mutagenesis canof course be carried out via PCR.

[0137] Enhancing Expression of Polymerases

[0138] To optimize expression of the polymerases of the presentinvention, inducible or constitutive promoters are well known and may beused to express high levels of a polymerase structural gene in arecombinant host. Similarly, high copy number vectors, well known in theart, may be used to achieve high levels of expression. Vectors having aninducible high copy number may also be useful to enhance expression ofthe polymerases of the invention in a recombinant host.

[0139] To express the desired structural gene in a prokaryotic cell(such as, E. coli, B. subtilis, Pseudomonas, etc.), it is necessary tooperably link the desired structural gene to a functional prokaryoticpromoter. However, the natural promoter of the polymerase gene mayfunction in prokaryotic hosts allowing expression of the polymerasegene. Thus, the natural promoter or other promoters may be used toexpress the polymerase gene. Such other promoters may be used to enhanceexpression and may either be constitutive or regulatable (i.e.,inducible or derepressible) promoters. Examples of constitutivepromoters include the int promoter of bacteriophage λ, and the blapromoter of the β-lactamase gene of pBR322. Examples of inducibleprokaryotic promoters include the major right and left promoters ofbacteriophage λ (P_(R) and P_(L)), trp, recA, lacZ, lacI, tet, gal, trc,and tac promoters of E. coli. The B. subtilis promoters includeα-amylase (Ulmanen et al., J. Bacteriol 162:176-182 (1985)) and Bacillusbacteriophage promoters (Gryczan, T., In: The Molecular Biology OfBacilli, Academic Press, New York (1982)). Streptomyces promoters aredescribed by Ward et al., Mol. Gen. Genet. 203:468478 (1986)).Prokaryotic promoters are also reviewed by Glick, J. Ind. Microbiol.1:277-282 (1987); Cenatiempto, Y., Biochimie 68:505-516 (1986); andGottesman, Ann. Rev. Genet. 18:415-442 (1984). Expression in aprokaryotic cell also requires the presence of a ribosomal binding siteupstream of the gene-encoding sequence. Such ribosomal binding sites aredisclosed, for example, by Gold et al., Ann. Rev. Microbiol. 35:365404(1981).

[0140] To enhance the expression of polymerases of the invention in aeukaryotic cell, well known eukaryotic promoters and hosts may be used.Preferably, however, enhanced expression of the polymerases isaccomplished in a prokaryotic host. The preferred prokaryotic host foroverexpressing the polymerases of the invention is E. coli.

[0141] Isolation and Purification of Polymerases

[0142] The enzyme(s) of the present invention is preferably produced byfermentation of the recombinant host containing and expressing thedesired polymerase gene. However, the polymerases of the presentinvention may be isolated from any strain which produces the polymeraseof the present invention. Fragments of the polymerase are also includedin the present invention. Such fragments include proteolytic fragmentsand fragments having polymerase activity.

[0143] Any nutrient that can be assimilated by a host containing thepolymerase gene may be added to the culture medium. Optimal cultureconditions should be selected case by case according to the strain usedand the composition of the culture medium. Antibiotics may also be addedto the growth media to insure maintenance of vector DNA containing thedesired gene to be expressed. Media formulations have been described inDSM or ATCC Catalogs and Sambrook et al., In: Molecular Cloning, aLaboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

[0144] Host cells producing the polymerases of this invention can beseparated from liquid culture, for example, by centrifugation. Ingeneral, the collected microbial cells are dispersed in a suitablebuffer, and then broken down by ultrasonic treatment or by other wellknown procedures to allow extraction of the enzymes by the buffersolution. After removal of cell debris by ultracentrifugation orcentrifugation, the polymerase can be purified by standard proteinpurification techniques such as extraction, precipitation,chromatography, affinity chromatography, electrophoresis or the like.Assays to detect the presence of the polymerase during purification arewell known in the art and can be used during conventional biochemicalpurification methods to determine the presence of these enzymes.

[0145] Thermotoga Polymerases

[0146] Thermotoga polymerases for use in the present invention areobtained from any strain of Thermotoga species, more preferably from astrain of Thermotoga neapolitana (WO 96/10640 or WO96/41014) orThermotoga maritima (U.S. Pat. No. 5,374,553). Enzymes suitable for usein the present invention from these more preferred sources are thewildtype DNA polymerases (Tne from T. neapolitana; Tma from T.maritima), or mutants or derivatives thereof

[0147] The present invention provides isolated nucleic acid moleculesencoding preferred mutant Tne DNA polymerases, mutant Tne DNApolymerases encoded by such isolated nucleic acid molecules, andspecific mutant Tne DNA polymerase proteins. Most preferred are thewildtype Tne DNA polymerase (SEQ ID NOs:1,2), the wildtype Tma DNApolymerase (U.S. Pat. No. 5,374,553), and the following mutants of TneDNA polymerase: Tne N′Δ219, D323A (SEQ ID NO:3); Tne N′Δ283, D323A (SEQID NO:4); Tne N′Δ192, D323A (SEQ ID NO:5); Tne D137A, D323A (SEQ IDNO:6); Tne D8A, D323A (SEQ ID NO:7); Tne G195D, D323A (SEQ ID NO:8); TneG37D, D323A (SEQ ID NO:9); Tne N′Δ283 (SEQ ID NO:10); Tne D137A, D323A,R722K; Tne D137A, D323A, R722Y; Tne D137A, D323A, R722L; Tne D137A,D323A, R722H; Tne D137A, D323A, R722Q; Tne D137A, D323A, F730Y; TneD137A, D323A, K726R; Tne D137A, D323A, K726H; Tne D137A, D323A, R722K,F730Y; Tne D137A, D323A, R722K, K726R; Tne D137A, D323A, R722K, K726H;Tne D137A, D323A, R722H, F730Y; Tne D137A, D323A, R722H, K726R; TneD137A, D323A, R722H, K726H; Tne D137A, D323A, R722Q, F730Y; Tne D137A,D323A, R722Q, K726R; Tne D137A, D323A, R722Q, K726H; Tne D137A, D323A,R722N, F730Y; Tne D137A, D323A, R722N, K726R; Tne D137A, D323A, R722N,K726H; Tne D137A, D323A, F730S; Tne N′Δ283, D323A, R722K/H/Q/N/Y/L; TneN′Δ219, D323A, R722K; Tne N′Δ219, D323A, F730Y; Tne N′Δ219, D323A,K726R; Tne N′Δ219, D323A, K726H; Tne D137A, D323A, F730S,R722K/Y/Q/N/H/L, K726R/H; Tne D137A, D323A, F730T, R722K/Y/Q/N/H/L,K726R/H; Tne D137A, D323A, F730T; Tne F730S; Tne F730A; Tne K726R; TneK726H; and Tne D137A, D323A, R722N. It will of course be understood bythe skilled artisan that the designations of the above-described mutantpolymerases indicate the position of the amino acid residue in thewildtype amino acid sequence (SEQ ID NO:2) that is being mutated, aswell as to what residue the amino acid is being mutated. Thus, forexample, “D137A” indicates that the Asp (D) residue at position 137 inSEQ ID NO:2 has been mutated to an Ala (A) residue, and, for example,“R722K/Y/Q/N/H/L” indicates that the Arg (R) residue at position 722 inSEQ ID NO:2 has been mutated to a Lys (K), Tyr (Y), Gln (Q), Asn (N),His (H) or Leu (L) residue. Mutant polymeraes having one or moremutations or modifications corresponding to the Tne mutants of theinvnetion are also contemplated by the invention.

[0148] The following chart indicates the nucleic acid sequences of thenucleic acid molecules encoding the above-described mutant Tne DNApolymerases (SEQ ID NOs:3-10), each with reference to the wildtype TneDNA polymerase (SEQ ID NO: 1): SEQ ID NO: Deletion of SEQ ID NO:1Insertion Substitution to SEQ ID NO:1 3 Deletion of positions 1- ATG AGCTTC A replaces G at 657 from the 5′-end at the 5′-end position 966; Creplaces A at 968 and G replaces C at 969 4 Deletion of positions 1-None A replaces G at 966; C 849 from the 5′-end replaces A at 968 and Greplaces C at 969 5 Deletion of positions 1- ATG AAT TCG A replaces G at966; C 576 from the 5′-end AGC TCG GTA replaces A at 968 and G CCC atthe 5′- replaces C at 969; A end replaces G at 584 6 None None Areplaces G at 966; C replaces A at 968 and G replaces C at 969; Creplaces T at 408 and C replaces A at 410 7 None None A replaces G at966; C replaces A at 968 and G replaces C at 969; C replaces A at 23 andC replaces T at 24 8 None None A replaces G at 966; C replaces A at 968and G replaces C at 969; T replaces C at 576 and A replaces G at 584 9None None A replaces G at 966; C replaces A at 968 and G replaces C at969; A replaces G at 110 10 Deletion of positions 1- None None 849 fromthe 5′-end

[0149] Using these same approaches, the sequence guidance providedherein, and knowledge of appropriate nucleotide substitutions to be madeto SEQ ID NO:1, one of ordinary skill can readily produce other nucleicacid molecules encoding mutant polymerases, such as those described indetail above, having the desired activity. In addition, other nucleicacid molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode a mutant Tne DNA polymerase having an amino acidsequence set forth above, are also encompassed by the present invention.Since the genetic code is well known in the art, it is routine for oneof ordinary skill in the art to produce such mutants and degeneratevariants without undue experimentation.

[0150] Each of these mutant Tne DNA polymerases are reduced orsubstantially reduced in the ability to add a non-templated 3′ terminalnucleotide to the growing strand. These mutant Tne DNA polymeraseproteins may be prepared by recombinant DNA techniques routine to one ofordinary skill. Preferably, such mutant Tne polymerases are prepared byinserting an isolated DNA molecule having a nucleotide sequence asdescribed above for each individual mutant into a recombinant vector,inserting the vector into a host cell, preferably an Escherichia colicell, and culturing the host cell under conditions favoring theproduction of the mutant Tne DNA polymerase. The mutant Tne polymeraseis then isolated from the host cell according to standard proteinpurification techniques. Further guidance for the preparation andisolation of mutant DNA polymerases from thermostable microorganisms canbe found, for example, in U.S. Pat. No. 5,374,553, in co-pending U.S.patent application Ser. No. 08/689,818 of Deb K. Chatterjee and A. JohnHughes, entitled “Cloned DNA Polymerases from Thermotoga and MutantsTherof,” filed Sep. 6, 1996, and in co-pending U.S. patent applicationSer. No. 08/689,807 of Deb K. Chatterjee, entitled “Cloned DNAPolymerases from Thermotoga and Mutants Therof,” filed Sep. 6, 1996, thedisclosures of all of which are incorporated herein in their entirety.

[0151] In the methods of the present invention, Thermotoga DNApolymerases substantially reduced in 3′-5′ exonuclease activity (such asa Tne mutant having an amino acid sequence as set forth in any one ofSEQ ID NOs:3-9), or Thermotoga DNA polymerases not substantially reducedin 3′-5′ exonuclease activity (such as Tne DNA polymerase (SEQ IDNOs:1,2), Tma DNA polymerase (U.S. Pat. No. 5,374,553), or the Tnemutant Tne N′Δ283 (SEQ ID NO:10)), may be used with similar results,since both types of Thermotoga DNA polymerase are substantially reducedin the ability to add a nontemplated 3′ terminal nucleotide to a DNAtemplate. Other thermostable DNA polymerases substantially reduced in3′-5′ exonuclease activity, such as Taq, VENT™(exo−), DEEPVENT™(exo−),Dtok(exo−) and THERMOLASE™ Tbr, are not preferred for use in the presentmethods as they will add non-templated nucleotides to the 3′ termini ofthe amplification products as described below. However, suchthermostable polymerase can be made which have reduced, substantiallyreduced or eliminated activity to add 3′ non-template nucleotides bymutating or modifying the polymerase in accordance with the invention.The preferred Thermotoga polymerases of the invention contain suchmutations or modifications in their O-helix.

[0152] The recombinant host comprising the gene encoding Tne DNApolymerase, E. coli DH10B(pUC-Tne), was deposited on Sep. 30, 1994, withthe Collection, Agricultural Research Culture Collection (NRRL), 1815North University Street, Peoria, Ill. 61604 USA, as Deposit No. NRRLB-21238. The gene encoding Tma DNA polymerase has also been cloned andsequenced (U.S. Pat. No. 5,374,553, which is expressly incorporated byreference herein in its entirety). Methods for preparing mutants andderivatives of these Tne and Tma polymerases are well-known in the art,and are specifically described in co-pending U.S. patent applicationSer. No. 08/689,818 of Deb K. Chatterjee and A. John Hughes, entitled“Cloned DNA Polymerases from Thermotoga and Mutants Therof,” filed Sep.6, 1996, and co-pending U.S. patent application Ser. No. 08/689,807 ofDeb K. Chatterjee, entitled “Cloned DNA Polymerases from Thermotoga andMutants Therof,” filed Sep. 6, 1996, the disclosures of which areincorporated herein in their entirety.

[0153] Advantages of Thermostable Polymerases

[0154] The use of thermostable polymerases (e.g. Thermotoga polymerases)or mutants or derivatives thereof in the methods of the presentinvention provide several distinct advantages. These advantages areparticularly apparent in the application of the present methods toanalysis and typing of minisatellite, microsatellite and STR DNAregions.

[0155] With respect to traditional thermolabile DNA polymerases used inDNA amplification and sequencing, such as T4, T7 or E. coli Klenowfragment polymerases, thermostable polymerases such as Thermotoga DNApolymerases maintain their enzymatic activity in the multiplehigh-temperature cycles used in PCR and analogous automatedamplification methodologies. It is therefore unnecessary to add freshenzyme at the beginning of each amplification cycle when usingthermostable polymerases, as must be done when thermolabile enzymes areused.

[0156] With respect to other thermostable enzymes, it has beenunexpectedly discovered in the present invention (as described in moredetail in the Examples below) that the use of Tne or Tma DNA polymerasemutants or derivatives thereof, does not result in the incorporation ofnon-templated 3′ nucleotides into the newly synthesized DNA strandsduring DNA amplification reactions. This non-templated incorporation isa common problem when using certain other commonly employed thermostableenzymes, such as Taq, VENT™(exo−), DEEPVENT™(exo−), Dtok(exo−) andTHERMOLASE™ Tbr. It has also been unexpectedly discovered that mutantsof these polymerases can be made to reduce or eliminate addition ofnon-templated 3′ nucleotides. In particular, such mutations arepreferably made within the O-helix of such polymerases.

[0157] Thus, the use of Tne or Tma DNA polymerases or mutants orderivatives thereof (or other mutant polymerases produced according tothe invention) in amplifying and typing DNA sequences, particularlyhypervariable DNA sequences such as minisatellite, microsatellite or STRregions, will allow a faithful amplification and resolution ofpolymorphisms in these regions. This faithful resolution is not possibleusing other thermostable polymerases due to their propensity fornon-templated incorporation. Thus, these enzymes are suitable for use inautomated amplification systems such as PCR.

[0158] Sources of DNA

[0159] Suitable sources of DNA, including a variety of cells, tissues,organs or organisms, may be obtained through any number of commercialsources (including American Type Culture Collection (ATCC), Rockville,Md.; Jackson Laboratories, Bar Harbor, Me.; Cell Systems, Inc.,Kirkland, Wash.; Advanced Tissue Sciences, La Jolla, Calif.). Cells thatmay be used as starting materials for genomic DNA preparation arepreferably eukaryotic (including fungi or yeasts, plants, protozoans andother parasites, and animals including humans and other mammals).Although any mammalian cell may be used for preparation of DNA,preferred are blood cells (erythrocytes and leukocytes), endothelialcells, epithelial cells, neuronal cells (from the central or peripheralnervous systems), muscle cells (including myocytes and myoblasts fromskeletal, smooth or cardiac muscle), connective tissue cells (includingfibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes andosteoblasts) and other stromal cells (e.g., macrophages, dendriticcells, Schwann cells), although other cells, including the progenitors,precursors and stem cells that give rise to the above-described somaticcells, are equally suitable. Also suitable for use in the preparation ofDNA are mammalian tissues or organs such as those derived from brain,kidney, liver, pancreas, blood, bone marrow, muscle, nervous, skin,genitourinary, circulatory, lymphoid, gastrointestinal and connectivetissue sources, as well as those derived from a mammalian (includinghuman) embryo or fetus. These cells, tissues and organs may be normal,or they may be pathological such as those involved in infectiousdiseases (caused by bacteria, fungi or yeast, viruses (including AIDS)or parasites), in genetic or biochemical pathologies (e.g., cysticfibrosis, hemophilia, Alzheimer's disease, schizophrenia, musculardystrophy or multiple sclerosis), or in cancerous processes.

[0160] More specifically, in one aspect of the invention, therelationship between a first individual and a second individual may bedetermined by analyzing and typing a particular polymorphic DNAfragment, such as a minisatellite or microsatellite DNA sequence. Insuch a method, the amplified fragments for each individual are comparedto determine similarities or dissimilarities. Such an analysis isaccomplished, for example, by comparing the size of the amplifiedfragments from each individual, or by comparing the sequence of theamplified fragments from each individual. In another aspect of theinvention, genetic identity can be determined. Such identity testing isimportant, for example, in paternity testing, forensic analysis, etc. Inthis aspect of the invention, a sample containing DNA (e.g., a crimescene sample or a sample from an individual) is analyzed and compared toa sample from one or more individuals. In one such aspect of theinvention, one sample of DNA may be derived from a first individual andanother sample may be derived from a second individual whoserelationship to the first individual is unknown; comparison of thesesamples from the first and second individuals by the methods of theinvention may then facilitate a determination of the genetic identity orrelationship between the first and second individual. In a particularlypreferred such aspect, the first DNA sample may be a known samplederived from a known individual and the second DNA sample may be anunknown sample derived, for example, from crime scene material. In anadditional aspect of the invention, one sample of DNA may be derivedfrom a first individual and another sample may be derived from a secondindividual who is related to the first individual; comparison of thesesamples from the first and second individuals by the methods of theinvention may then facilitate a determination of the genetic kinship ofthe first and second individuals by allowing examination of theMendelian inheritance, for example, of a polymorphic, minisatellite,microsatellite or STR DNA fragment. In another aspect of the invention,DNA fragments important as genetic markers for encoding a gene ofinterest can be identified and isolated. For example, by comparingsamples from different sources, DNA fragments which may be important incausing diseases such as infectious diseases (of bacterial, fungal,parasitic or viral etiology), cancers or genetic diseases, can beidentified and characterized. In this aspect of the invention a DNAsample from normal cells or tissue is compared to a DNA sample fromdiseased cells or tissue. Upon comparison according to the invention,one or more unique polymorphic fragments present in one DNA sample andnot present in the other DNA sample can be identified and isolated.Identification of such unique polymorphic fragments allows foridentification of sequences associated with, or involved in, causing thediseased state.

[0161] Once the starting cells, tissues, organs or other samples areobtained, DNA may be prepared therefrom by methods that are well-knownin the art (See, e.g., Maniatis, T., et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press, pp. 9.16-9.23 (1989); Kaufman, P. B., et al., Handbookof Molecular and Cellular Methods in Biology and Medicine, Boca Raton,Fla.: CRC Press, pp. 1-26 (1995)). The DNA samples thus prepared maythen be used to identify, analyze and type polymorphic DNA fragments,including minisatellite, microsatellite and STR DNA fragments, byamplification, preferably by PCR amplification, as modified by themethods of the present invention.

[0162] General methods for amplification and analysis of DNA fragmentsare well-known to one of ordinary skill in the art (see, e.g., U.S. Pat.Nos. 4,683,195; 4,683,202; and 4,800,159; Innis, M. A., et al., eds.,PCR Protocols: A Guide to Methods and Applications, San Diego, Calif.:Academic Press, Inc. (1990); Griffin, H. G., and Griffin, A. M., eds.,PCR Technology: Current Innovations, Boca Raton, Fla.: CRC Press(1994)). Typically, these methods comprise contacting the DNA samplewith a thermostable DNA polymerase in the presence of one or more primersequences, amplifying the DNA sample to generate a collection ofamplified polymorphic, minisatellite, microsatellite or STR DNAfragments, preferably by PCR or equivalent automated amplificationtechnique, separating the amplified DNA fragments by size, preferably bygel electrophoresis, and analyzing the gels for the presence ofpolymorphic, minisatellite, microsatellite or STR DNA fragments bydirect comparison of the pattern of fragments generated from a firstsample of DNA to those from a second sample of DNA, or by a moreindirect comparison using known size markers.

[0163] As noted above; amplification protocols used heretofore foranalyzing and typing polymorphic DNA fragments, particularlyminisatellite, microsatellite or STR DNA sequences, use certainthermostable DNA polymerases such as Taq (U.S. Pat. Nos. 4,683,195;4,683,202; and 4,800,159). However, as discussed in detail above, theseapproaches yield amplification products in which one or morenon-templated nucleotides is added to the 3′ termini of the products bythe polymerases, thus leading to heterogeneity in the amplificationproducts, and ambiguity concerning the correct size of the amplificationproducts.

[0164] This problem is overcome in the present invention by contactingthe DNA sample in the amplification reaction mixtures with one or moreDNA polymerases of the invention which are reduced, substantiallyreduced or eliminated in the ability to add a nontemplated 3′ terminalnucleotide to the growing strand. Preferably, such DNA polymerases areThermotoga DNA polymerases, more preferably a Thermotoga DNA polymerasesubstantially reduced in 3′-5′ exonuclease activity, still morepreferably a Tne polymerase (SEQ ID NOs:1,2), a Tma polymerase (U.S.Pat. No. 5,374,553), or a mutant or derivative thereof, and mostpreferably one of the following mutants of Tne polymerase: Tne N′Δ219,D323A (SEQ ID NO:3); Tne N′Δ283, D323A (SEQ ID NO:4); Tne N′Δ192, D323A(SEQ ID NO:5); Tne D137A, D323A (SEQ ID NO:6); Tne D8A, D323A (SEQ IDNO:7); Tne G195D, D323A (SEQ ID NO:8); Tne G37D, D323A (SEQ ID NO:9);Tne N′A283 (SEQ ID NO:10); Tne D137A, D323A, R722K; Tne D137A, D323A,R722Y; Tne D137A, D323A, R722L; Tne D137A, D323A, R722H; Tne D137A,D323A, R722Q; Tne D137A, D323A, F730Y; Tne D137A, D323A, K726R; TneD137A, D323A, K726H; Tne D137A, D323A, R722K, F730Y; Tne D137A, D323A,R722K, K726R; Tne D137A, D323A, R722K, K726H; Tne D137A, D323A, R722H,F730Y; Tne D137A, D323A, R722H, K726R; Tne D137A, D323A, R722H, K726H;Tne D137A, D323A, R722Q, F730Y; Tne D137A, D323A, R722Q, K726R; TneD137A, D323A, R722Q, K726H; Tne D137A, D323A, R722N, F730Y; Tne D137A,D323A, R722N, K726R; Tne D137A, D323A, R722N, K726H; Tne D137A, D323A,F730S; Tne N′Δ283, D323A, R722K/H/Q/N/Y/L; Tne N′Δ219, D323A, R722K; TneN′Δ219, D323A, F730Y; Tne N′Δ219, D323A, K726R; Tne N′Δ219, D323A,K726H; Tne D137A, D323A, F730S, R722K/Y/Q/N/H/L, K726R/H; Tne D137A,D323A, F730T, R722K/Y/Q/N/H/L, K726R/H; Tne D137A, D323A, F730T; TneF730S; Tne F730A; Tne K726R; Tne K726H; and Tne D137A, D323A, R722N.

[0165] It will be understood, however, that other thermostable DNApolymerases or mutants thereof, any of which are reduced, substantiallyreduced, or eliminated in the ability to add a non-templated 3′ terminalnucleotide to the growing strand, may be used in the methods of thepresent invention equivalently. The DNA polymerases are used in themethods of the present invention at a concentration of about 0.0001units/ml to about 10 units/ml, preferably at a concentration of about0.001 units/ml to about 5 units/ml, more preferably at a concentrationof about 0.004 units/ml to about 1 unit/ml, and most preferably at aconcentration of about 0.04 units/ml. Thus, the methods of the presentinvention produce a population of amplified DNA fragments, mostpreferably of polymorphic or microsatellite DNA fragments, whichcomprise substantially no non-templated 3′ terminal nucleotides. By“substantially no non-templated 3′ terminal nucleotides” is meant thatthe population of amplified DNA fragments demonstrates about 0-50%,about 0-30%, about 0-20%, preferably about 0-10%, more preferably about0-5%, still more preferably about 0-1% and most preferably about 0%, ofDNA molecules containing non-templated 3′ nucleotides compared toamplified DNA fragments produced by the polymerase control. When testingthe ability of a DNA polymerase to add 3′ non-templated nucleotides, thepolymerase, when it has substantially reduced or eliminated 3′exonuclease activity, is compared to Taq DNA polymerase (see above).When testing polymerases which have been modified or mutated to reduceor eliminate 3′ non-templated nucleotide addition, the mutated ormodified polymerase is compared to the corresponding wildtype,unmodified or unmutated polymerase (see above).

[0166] Following amplification by the methods of the present invention,the amplified DNA fragments may be analyzed to identify or type apolymorphic, minisatellite, microsatellite or STR DNA fragment. Thisstep is usually accomplished by separation of the amplified DNAfragments by size, a procedure which permits the determination of thepresence of unique polymorphic fragments in one or more of the DNAsamples. The fragments may be separated by any physical or biochemicalmeans including gel electrophoresis, capillary electrophoresis,chromatography (including sizing, affinity and immunochromatography),density gradient centrifugation and immunoadsorption. For carrying outthe present invention, separation of DNA fragments by gelelectrophoresis is particularly preferred, as it provides a rapid andhighly reproducible means of sensitive separation of a multitude of DNAfragments, and permits direct, simultaneous comparison of the fragmentsin several samples of DNA, or samples of DNA from a first and a secondindividual.

[0167] Gel electrophoresis is typically performed on agarose orpolyacrylamide sequencing gels according to standard protocols,preferably using gels containing polyacrylamide at concentrations of3-12% and most preferably at about 8%, and containing urea at aconcentration of about 4-12M, most preferably about 8M. Samples areloaded onto the gels, usually with samples containing amplified DNAfragments prepared from different sources of genomic DNA being loadedinto adjacent lanes of the gel to facilitate subsequent comparison.Reference markers of known sizes may be used to facilitate thecomparison of samples. Following electrophoretic separation, DNAfragments may be visualized and identified by a variety of techniquesthat are routine to those of ordinary skill in the art, such asautoradiography. One can then examine the autoradiographic films eitherfor differences in polymorphic fragment patterns (“typing”) or for thepresence of one or more unique bands in one lane of the gel(“identifying”); the presence of a band in one lane (corresponding to asingle sample, cell or tissue type) that is not observed in other lanesindicates that the DNA fragment comprising that unique band issource-specific and thus a potential polymorphic DNA fragment.

[0168] A variety of DNA fragments comprising polymorphic, minisatellite,microsatellite or STR DNA fragments can thus be identified using themethods of the present invention by comparing the pattern of bands onthe films depicting various samples. Importantly, using the presentmethods the amplification products of the polymorphic DNA fragments willbe faithful copies of the template (allele) material—i.e., they will notexhibit undesired additional nucleotides at their 3′ termini vianon-templated addition of nucleotides by the polymerases. One can extendthis approach, in another preferred embodiment, to isolate andcharacterize these fragments or any DNA fragment amplified without thenon-templated addition of a 3′ terminal nucleotide. In this embodiment,one or more of the unique DNA fragments are removed from the gel whichwas used for identification (see above), according to standardtechniques such as electroelution or physical excision.

[0169] The isolated unique DNA fragments may then be inserted intostandard nucleotide vectors, including expression vectors, suitable fortransfection or transformation of a variety of prokaryotic (bacterial)or eukaryotic (yeast, plant or animal including human and othermammalian) cells. In particular, the present invention provides methodsof cloning such isolated unique DNA fragments, or any PCR-amplified DNAfragment, by blunt-end cloning. As described above, Taq DNA polymeraseadds a non-templated nucleotide, typically a deoxyadenosine (“A”), tothe 3′ terminus of the amplified DNA fragment. Thus, Taq-catalyzed PCRgenerates a collection of DNA fragments with 3′ A overhangs. To clonesuch Taq-amplified fragments, two approaches are commonly used: eitherthe 3′ A overhang is removed by treating the amplified fragment with,for example, T4 DNA polymerase (a technique sometimes called “3′polishing”), or a special cloning vector with a 3′T overhang (a “TAcloning vector”) is used. Of course, such approaches are moretime-consuming and expensive than if direct insertion of the amplifiedfragment were done. Such a direct approach is possible using the methodsof the invention, which generates little or no 3′ A overhangs (and thus,blunt ends) on the amplified DNA fragments. The DNA fragments, amplifiedaccording to the methods of the invention, may thus be directly insertedinto corresponding blunt-ended vectors according to standard techniques(for example, using T4 DNA ligase). Thus, the present invention providesa method of blunt-end cloning of a DNA fragment that obviates the use ofTA cloning vectors or 3′ polishing.

[0170] To identify the presence of minisatellite DNA fragments, thepolymorphic DNA fragments that are identified and isolated by themethods of the present invention may be further characterized, forexample by sequencing (i.e., determining the nucleotide sequence of thepolymorphic fragments), by methods described above and others that arestandard in the art (see, e.g., U.S. Pat. Nos. 4,962,022 and 5,498,523,which are directed to methods of DNA sequencing).

[0171] Kits

[0172] The invention also provides kits for use in the identification,analysis and typing of a polymorphic DNA fragment, particularly aminisatellite or STR DNA fragment, according to the present methods.Kits according to the present invention may comprise a carrying meansbeing compartmentalized to receive in close confinement therein one ormore containers such as vials, tubes, bottles and the like. Each of suchcontainers may comprise components or a mixture of components needed toperform DNA amplification or analysis.

[0173] Such kits may comprise of one or more thermostable DNApolymerases reduced, substantially reduced or eliminated in the abilityto add a non-templated 3′ nucleotide to a growing DNA strand. Preferablythe container contains a Thermotoga DNA polymerase or a mutant or aderivative thereof, particularly those described in full detail above.The kit may also contain one or more DNA primer molecules, one or moredeoxyribonucleoside triphosphates needed to synthesize a DNA moleculecomplementary to a DNA template, and/or a buffer suitable foramplification of a nucleic acid molecule (or combinations threof).

[0174] A kit for DNA analysis may include one or more of the abovecomponents, and may further include containers which contain reagentsnecessary for separation and analysis of DNA fragments, such aspolyacrylamide, agarose, urea, detergents and the like.

[0175] Of course, it is also possible to combine one or more of thesereagents in a single tube. A detailed description of such formulationsat working concentrations is described in co-pending U.S. applicationSer. No. 08/689,815 of Ayoub Rashtchian and Joseph Solus, entitled“Stable Compositions for Nucleic Acid Amplification and Sequencing”filed on Aug. 14, 1996, the disclosure of which is incorporated byreference herein in its entirety.

[0176] The invention also relates to kits for detectably labelingmolecules, sequencing, amplifying and synthesizing molecules by wellknown techniques. See U.S. Pat. Nos. 4,962,020, 5,173,411, 4,795,699,5,498,523, 5,405,776 and 5,244,797. Such kits may comprise a carryingmeans being compartmentalized to receive in close confinement one ormore container means such as vials, test tubes and the like. Each ofsuch container means comprises components or a mixture of componentsneeded to perform nucleic acid synthesis, sequencing, labeling, oramplification.

[0177] A kit for sequencing DNA may comprise a number of containermeans. Such a kit may comprise one or more of the polymerases of theinvention, one or a number of types of nucleotides needed to synthesizea DNA molecule complementary to DNA template, one or a number ofdifferent types of terminators (such as dideoxynucleosidetriphosphates), a pyrophosphatase, one or a number of primers and/or asuitable sequencing buffer (or combinations of such components).

[0178] A kit used for amplifying or synthesizing of nucleic acids willcomprise, one or more polymerases of the invention, and one or a numberof nucleotides or mixtures of nucleotides. Various primers may beincluded in a kit as well as a suitable amplification or synthesisbuffers.

[0179] When desired, the kit of the present invention may also includecontainer means which comprise detectably labeled nucleotides which maybe used during the synthesis or sequencing of a nucleic acid molecule.One of a number of labels may be used to detect such nucleotides.Illustrative labels include, but are not limited to, radioactiveisotopes, fluorescent labels, chemiluminescent labels, bioluminescentlabels and enzyme labels.

[0180] Use of the Methods and Kits

[0181] The polymeraes, methods and kits embodied in the presentinvention will have general utility in any application utilizing nucleicacid amplification methodologies, particularly those directed to theanalysis and typing of polymorphic or minisatellite DNA fragments, andmost particularly those directed to the analysis and typing ofminisatellite, microsatellite and STR DNA fragments. Amplificationtechniques in which the present methods may be used include PCR (U.S.Pat. Nos. 4,683,195 and 4,683,202), Strand Displacement Amplification(SDA; U.S. Pat. No. 5,455,166; EP 0 684 315), and Nucleic AcidSequence-Based Amplification (NASBA; U.S. Pat. No. 5,409,818; EP 0 329822). Nucleic acid analysis and typing techniques which may employ thepresent compositions include nucleic acid sequencing methods such asthose disclosed in U.S. Pat. Nos. 4,962,022 and 5,498,523, as well asmore complex PCR-based nucleic acid fingerprinting techniques such asRandom Amplified Polymorphic DNA (RAPD) analysis (Williams, J. G. K., etal., Nucl Acids Res. 18(22):653 1-6535, 1990), Arbitrarily Primed PCR(AP-PCR; Welsh, J., and McClelland, M., Nucl. Acids Res.18(24):7213-7218, 1990), DNA Amplification Fingerprinting (DAF;Caetano-Anolles et al., Bio/Technology 9:553-557, 1991), andmicrosatellite PCR or Directed Amplification of Minisatellite-region DNA(DAMD; Heath, D. D., et al., Nucl. Acids Res. 21(24): 5782-5785, 1993).In particular, the polymerases, methods and kits of the presentinvention will be useful in the fields of medical genetics, therapeuticsand diagnostics, forensics (particularly identity and paternitytesting), and agricultural (e.g., plant breeding) and other biologicalsciences, in any procedure utilizing DNA polymerases for analysis andtyping of polymorphic, minisatellite, microsatellite or STR DNAfragments. Particularly suitable for diagnosis by the methods of thepresent invention are genetic diseases such as cystic fibrosis,hemophilia, Alzheimer's disease, schizophrenia, muscular dystrophy ormultiple sclerosis. Together, these abilities will assist medicalprofessionals and patients in diagnostic and prognostic determinationsas well as in the development of treatment and prevention regimens forthese and other disorders.

[0182] It will also be apparent to one of ordinary skill in the art thatthe present methods may be used to screen animal tissues to besubsequently used in medical procedures such as tissue or organtransplants, blood transfusions, zygote implantations and artificialinseminations. In such procedures, pre-screening of the subject tissuesfor the presence of particular polymorphic DNA fragments may improve thesuccess of tissue or organ transplants (by decreasing the likelihood ofrejection due to donor-recipient genetic incompatibility) and of zygoteimplantations (by eliminating the use of genetically defective zygotes).Similarly, use of these methods will reduce the chances of transmissionof infectious diseases (e.g., hepatitis and AIDS) in medical proceduresthat are often prone to such transmission, such as blood transfusionsand artificial insemination. Finally, use of the present invention foridentification of unique polymorphic, minisatellite, microsatelliet andSTR DNA fragments will assist in forensic science in such applicationsas crime-scene analysis of blood, tissue and body secretions containingsmall amounts of DNA, as well as in paternity testing.

[0183] It will be readily apparent to those skilled in the relevant artsthat other suitable modifications and adaptations to the methods andapplications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereofHaving now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLES 1

[0184] Bacterial Strains and Growth Conditions

[0185]Thermotoga neapolitana DSM No. 5068 was grown under anaerobicconditions as described in the DSM catalog (addition of resazurin, Na₂S,and sulfur granules while sparging the media with nitrogen) at 85° C. inan oil bath from 12 to 24 hours. The cells were harvested by filteringthe broth through Whatman #1 filter paper. The supernatant was collectedin an ice bath and then centrifuged in a refrigerated centrifuge at8,000 rpms for twenty minutes. The cell paste was stored at −70° C.prior to total genomic DNA isolation.

[0186]E. coli strains were grown in 2×LB broth base (Lennox L brothbase: GIBCO/BRL) medium. Transformed cells were incubated in SOC (2%tryptone, 0.5% yeast extract, yeast 10 mM NaCl, 2.5 mM KCl, 20 mMglucose, 10 mM MgCl₂, and 10 mM MgSO₄ per liter) before plating. Whenappropriate antibiotic supplements were 20 mg/l tetracycline and 100mg/l ampicillin. E. coli strain DH10B (Lorow et al., Focus 12:19-20(1990)) was used as host strain. Competent DH10B may be obtained fromLife Technologies, Inc. (LTI) (Rockville, Md.).

EXAMPLE 2

[0187] DNA Isolation

[0188]Thermotoga neapolitana chromosomal DNA was isolated from 1.1 g ofcells by suspending the cells in 2.5 ml TNE (5 mM Tris-HCl, pH 8.0, 50mM NaCl, 10 mM EDTA) and treated with 1% SDS for 10 minutes at 37° C.DNA was extracted with phenol by gently rocking the lysed cellsovernight at 4° C. The next day, the lysed cells were extracted withchloroform:isoamyl alcohol. The resulting chromosomal DNA was furtherpurified by centrifugation in a CsCl density gradient. Chromosomal DNAisolated from the density gradient was extracted three times withisopropanol and dialyzed overnight against a buffer containing 10 mMTris-HCl (pH 8.0) and 1 mM EDTA (TE).

EXAMPLE 3

[0189] Construction of Genomic Libraries

[0190] The chromosomal DNA isolated in Example 2 was used to construct agenomic library in the plasmid pCP13. Briefly, 10 tubes each containing10 μg of Thermotoga neapolitana chromosomal DNA was digested with 0.01to 10 units of SauIIIAl for 1 hour at 37° C. A portion of the digestedDNA was tested in an agarose (1.2%) gel to determine the extent ofdigestion. Samples with less than 50% digestion were pooled, ethanolprecipitated and dissolved in TE. 6.5 μg of partially digestedchromosomal DNA was ligated into 1.5 μg of pCP13 cosmid which had beendigested with BamHI restriction endonuclease and dephosphorylated withcalf intestinal alkaline phosphatase. Ligation of the partially digestedThermotoga DNA and BamHI cleaved pCP13 was carried out with T4 DNAligase at 22° C. for 16 hours. After ligation, about 1 μg of ligated DNAwas packaged using λ-packaging extract (obtained from Life Technologies,Inc., Rockville, Md.). DH10B cells (Life Tech. Inc.) were then infectedwith 100 μl of the packaged material. The infected cells were plated ontetracycline containing plates. Serial dilutions were made so thatapproximately 200 to 300 tetracycline resistant colonies were obtainedper plate.

EXAMPLE 4

[0191] Screening for Clones Expressing Thermotoga neapolitana DNAPolymerase

[0192] Identification of the Thermotoga neapolitana DNA polymerase geneof the invention was cloned using the method of Sagner et al., Gene97:119-123 (1991) which reference is herein incorporated in itsentirety. Briefly, the E. coli tetracycline resistant colonies fromExample 3 were transferred to nitrocellulose membranes and allowed togrow for 12 hours. The cells were then lysed with the fumes ofchloroform:toluene (1:1) for 20 minutes and dried for 10 minutes at roomtemperature. The membranes were then treated at 95° C. for 5 minutes toinactivate the endogenous E. coli enzymes. Surviving DNA polymeraseactivity was detected by submerging the membranes in 15 ml of polymerasereaction mix (50 mM Tris-HCl (pH 8.8), 1 MM MgCl₂, 3 mMβ-mercaptoethanol, 10 μM dCTP, dGTP, dTTP, and 15 μCi of 3,000 Ci/mmol[α³²P]dATP) for 30 minutes at 65° C.

[0193] Using autoradiography, three colonies were identified thatexpressed a Thermotoga neapolitana DNA polymerase. The cells were grownin liquid culture and the protein extract was made by sonication. Thepresence of the cloned thermostable polymerase was confirmed bytreatment at 90° C. followed by measurement of DNA polymerase activityat 72° C. by incorporation of radioactive deoxyribonucleosidetriphosphates into acid insoluble DNA. One of the clones, expressing TneDNA polymerase, contained a plasmid designated pCP13-32 and was used forfurther study.

EXAMPLE 5

[0194] Subcloning of Tne DNA polymerase

[0195] Since the pCP13-32 clone expressing the Tne DNA polymerase genecontains about 25 kb of T. neapolitana DNA, subcloning a smallerfragment of the Tne polymerase gene was attempted. The molecular weightof the Tne DNA polymerase purified from E. coli/pCP13-32 was about 100kd. Therefore, a 2.5-3.0 kb DNA fragment will be sufficient to code forfull-length polymerase. A second round of Sau3A partial digestionsimilar to Example 3 was done using pCP13-32 DNA. In this case, a 3.5 kbregion was cut out from the agarose gel, purified by Gene Clean (BIO101, La Jolla, Calif.) and ligated into plasmid pSport 1 (LifeTechnologies, Inc.) which had been linearized with BamHI anddephosphorylated with calfintestinal alkaline phosphatase. Afterligation, DH10B was transformed and colonies were tested for DNApolymerase activity as described in Example 1. Several clones wereidentified that expressed Tne DNA polymerase. One of the clones(pSport-Tne) containing about 3 kb insert was further characterized. Arestriction map of the DNA fragment is shown in FIG. 1. Further, a 2.7Kb HindIII-SstI fragment was subcloned into pUC19 to generate pUC19-Tne. E. coli/pUC19-Tne also produced Tne DNA polymerase. E. coliDH10B (pUC19-Tne) was deposited on Sept. 30, 1994 with the Collection,Agricultural Research Culture Collection (NRRL), 1815 Peoria, Ill. 61604as Deposit No. NRRL B-21338. The nucleotide and amino acid sequence ofTne polymerase is described in U.S. application Ser. Nos. 08/706,702 and08/706,706 filed Sep. 9, 1996, both of which are incorporated byreference herein.

EXAMPLE 6

[0196] Purification of Thermotoga neapolitana DNA Polymerase from E.coli

[0197] Twelve grams of E. coli cells expressing cloned Tne DNApolymerase (DH10B/pSport-Tne) were lysed by sonication (fourthirty-second bursts with a medium tip at the setting of nine with aHeat Systems Ultrasonics Inc., model 375 sonicator) in 20 ml of ice coldextraction buffer (50 mM Tris HCl (pH 7.4), 8% glycerol, 5 mMmercaptoethanol, 10 mM NaCl, 1 mM EDTA, 0.5 mM PMSF). The sonicatedextract was heated at 80° C. for 15 min. and then cooled in ice for 5min. 50 mM KCl and PEI (0.4%) was added to remove nucleic acids. Theextract was centrifuged for clarification. Ammonium sulfate was added to60%, the pellet was collected by centrifugation and resuspended in 10 mlof column buffer (25 mM Tris-HCl (pH 7.4), 8% glycerol, 0.5% EDTA, 5 mM2-mercaptoethanol, 10 mM KCl). A Blue-Sepharose (Pharmacia) column, orpreferably a Toso heparin (Tosohaas) column, was washed with 7 columnvolumes of column buffer and eluted with a 15 column volume gradient ofbuffer from 10 mM to 2 M KCl. Fractions containing polymerase activitywere pooled. The fractions were dialyzed against 20 volumes of columnbuffer. The pooled fractions were applied to a Toso650Q column(Tosohaas). The column was washed to baseline OD₂₈₀ and elution effectedwith a linear 10 column volume gradient of 25 mM Tris (pH 7.4), 8%glycerol, 0.5 mM EDTA, 10 mM KCl, 5 mM β-mercaptoethanol to the samebuffer plus 650 mM KCl. Active fractions were pooled.

EXAMPLE 7

[0198] Construction of Thermotoga neapolitana 3′-to-5′ ExonucleaseMutant

[0199] The amino acid sequence of portions of the Tne DNA polymerase wascompared with other known DNA polymerases such as E. coli DNA polymerase1, Taq DNA polymerase, T5 DNA polymerase, and T7 DNA polymerase tolocalize the regions of 3′-to-5′ exonuclease activity, and the dNTPbinding domains within the DNA polymerase. One of the 3′-to-5′exonuclease domains was determined based on the comparison of the aminoacid sequences of various DNA polymerases (Blanco, L., et al. Gene 112:139-144 (1992); Braithwaite and Ito, Nucleic Acids Res. 21: 787-802(1993)) is as follows: Tne 318 PSFALDLETSS 328 (SEQ ID NO:18) Po1 I 350PVFAFDTETDS 360 (SEQ ID NO:19) T5 159 GPVAFDSETSA 169 (SEQ ID NO:20) T71 MIVSDIEANA 10 (SEQ ID NO:21)

[0200] As a first step to make the Tne DNA polymerase devoid of 3′→5′exonuclease activity, a 2 kb Sph fragment from pSport-Tne was clonedinto M13mp19 (LTI, Rockville, Md.). The recombinant clone was selectedin E. coli DH5αF′IQ (LTI, Rockville, Md.). One of the clones with theproper insert was used to isolate uracilated single-stranded DNA byinfecting E. coli CJ236 (Biorad, California) with the phage particleobtained from E. coli DH5αF′IQ. An oligonucleotide, GA CGT TTC AAG CGCTAG GGC AAA AGA (SEQ ID NO:22) was used to perform site directedmutagenesis. This site-directed mutagenesis converted Asp³²³ (indicatedas * above) to Ala³²³. An Eco47III restriction site was created as partof this mutagenesis to facilitate screening of the mutant followingmutagenesis. The mutagenesis was performed using a protocol as describedin the Biorad manual (1987) except T7 DNA polymerase was used instead ofT4 DNA polymerase (USB, Cleveland, Ohio). The mutant clones werescreened for the Eco47III restriction site that was created in themutagenic oligonucleotide. One of the mutants having the createdEco47III restriction site was used for further study. The mutationAsp³²³ to Ala³²³ was confirmed by DNA sequencing.

[0201] To incorporate the 3′→5′ exonuclease mutation in an expressionvector, the mutant phage was digested with SphI and HindIII. A 2 kbfragment containing the mutation was isolated. This fragment was clonedin pUC-Tne to replace the wild type fragment. See FIG. 2A. The desiredclone, pUC-Tne (3′→5′), was isolated. The presence of the mutantsequence was confirmed by the presence of the unique Eco47III site. Theplasmid was then digested with SstI and HindIII. The entire mutantpolymerase gene (2.6 kb) was purified and cloned into SstI and HindIIIdigested pTrc99 expression vector (Pharmacia, Sweden). The clones wereselected in DH10B (LTI, Rockville, Md.). The resulting plasmid wasdesignated pTrcTne35. See FIG. 2B. This clone produced active heatstable DNA polymerase.

EXAMPLE 8

[0202] Phenylalanine to Tyrosine Mutant

[0203] The polymerase active site including the dNTP binding domain isusually present at the carboxyl terminal region of the polymerase. Thesequence of the Tne polymerase gene suggests that the amino acids thatpresumably contact and interact with the dNTPs are present within the694 bases starting at the internal BamHI site. See FIG. 1. Thisconclusion is based on homology with a prototype polymerase E. coli DNApolymerase I. See Polisky et al., J. Biol. Chem. 265:14579-14591 (1990).A comparison was made of the O-helix for various polymerases: Tne 722RRVGKMVNFSIIYG 735 (SEQ ID NO:12) Po1 I 754 RRSAKAINFGLIYG 767 (SEQ IDNO:13) T5 562 RQAAKAITFGILYG 575 (SEQ ID NO:14) T7 518 RDNAKTFIYGFLYG531 (SEQ ID NO:15) Taq 659 RRAAKTINFGVLYG 672 (SEQ ID NO:16)

[0204] In order to change Phe⁷³⁰ of the Tne polymerase to a Tyr⁷³⁰ sitedirected mutagenesis was performed using the oligonucleotide GTA TAT TATAGA GTA GTT AAC CAT CTT TCC A (SEQ ID NO:23). As part of thisoligonucleotide directed mutagenesis, a HpaI restriction site wascreated in order to screen mutants easily. The same uracilatedsingle-stranded DNA and mutagenesis procedure described in Example 7were used for this mutagenesis. Following mutagenesis, the mutants werescreened for the HpaI site. Mutants with the desired HpaI site were usedfor further study. The mutation was confirmed by DNA sequencing.

[0205] The Phe⁷³⁰ to Tyr⁷³⁰ mutation was incorporated into pUC-Tne byreplacing the wild type SphI -HindIII fragment with the mutant fragmentobtained from the mutant phage DNA. The presence of the desired clone,pUC-TneFY, was confirmed by the presence of the unique HpaI site, seeFIG. 2A. The entire mutant polymerase gene was subcloned into pTrc99 asan SstI-HindIII fragment as described above in DH10B. The resultingplasmid was designated pTrcTneFY. (FIG. 2B). The clone produced activeheat stable polymerase.

EXAMPLE 9

[0206] 3′→5′ Exonuclease and Phe⁷³⁰→Tyr⁷³⁰ Double Mutants

[0207] In order to introduce the 3′→5′ exonuclease mutation and thePhe⁷³⁰→Tyr⁷³⁰ mutation in the same expression vector, pTrc99, it wasnecessary to first reconstitute both mutations in the pUC-Tne clone. SeeFIG. 3. Both the pUC-Tne (3′→5′) and the pUC-TneFY were digested withBamHI. The digested pUC-Tne (3′→5′) was dephosphorylated to avoidrecirculation in the following ligations. The resulting fragments werepurified on a 1% agarose gel. The largest BamHI fragment (4.4 kb) waspurified from pUC-Tne (3′→5′) digested DNA and the smallest BamHIfragment (0.8 kb) containing the Phe⁷³⁰→Tyr⁷³⁰ mutation was purified andligated to generate pUC-Tne35FY. The proper orientation and the presenceof both mutations in the same plasmid was confirmed by Eco47III, HpaI,and SphI-HindIII restriction digests. See FIG. 3.

[0208] The entire polymerase containing both mutations was subcloned asa SstI-HindIII fragment in pTrc99 to generate pTrcTne35FY in DH10B. Theclone produced active heat stable polymerase.

EXAMPLE 10

[0209]3′→5′ Exonuclease, 5′→3′ Exonuclease, and Phe⁷³⁰→Tyr⁷³⁰ TripleMutants

[0210] In most of the known polymerases, the 5′-to-3′ exonucleaseactivity is present at the amino terminal region of the polymerase(Ollis, D. L., et al., Nature 313, 762-766, 1985; Freemont, P. S., etal., Proteins 1, 66-73, 1986; Joyce, C. M., Curr. Opin. Struct. Biol. 1:123-129 (1991). There are some conserved amino acids that are implicatedto be responsible for 5′-to-3′ exonuclease activity (Gutman and Minton,Nucl. Acids Res. 21, 4406-4407, 1993). See supra. It is known that5′-to-3′ exonuclease domain is dispensable. The best known example isthe Klenow fragment of E. coli Pol I. The Klenow fragment is a naturalproteolytic fragment devoid of 5′-to-3′ exonuclease activity (Joyce, C.M., et al., J. Biol. Chem. 257, 1958-1964, 1990). In order to generatean equivalent mutant for Tne DNA polymerase devoid of 5′-to-3′exonuclease activity, the presence of a unique SphI site present 680bases from the SstI site was exploited. pUC-Tne35FY was digested withHindIII, filled-in with Klenow fragment to generate a blunt-end, anddigested with SphI. The 1.9 kb fragment was cloned into an expressionvector pTTQ19 (Stark, M. J. R., Gene 51,255-267, 1987) at the SphI-SmaIsites and was introduced into DH10B. This cloning strategy generated anin-frame polymerase clone with an initiation codon for methionine fromthe vector. The resulting clone is devoid of 219 amino terminal aminoacids of Tne DNA polymerase. This clone is designated as pTTQTne535FY(FIG. 4). The clone produced active heat stable polymerase. Noexonuclease activity could be detected in the mutant polymerase asevidenced by lack of presence of unusual sequence ladders in thesequencing reaction. This particular mutant polymerase is highlysuitable for DNA sequencing.

EXAMPLE 11

[0211]5′→3′ Exonuclease Deletion and Phe⁷³⁰→Tyr⁷³⁰ Substitution Mutant

[0212] In order to generate the 5′-to-3′ exonuclease deletion mutant ofthe Tne DNA polymerase Phe⁷³⁰→Tyr⁷³⁰ mutant, the 1.8 kb SphI-SpeIfragment of pTTQTne535FY was replaced with the identical fragment ofpUC-Tne FY. See FIG. 4. A resulting clone, pTTQTne5FY, produced activeheat stable DNA polymerase. As measured by the rate of degradation of alabeled primer, this mutant has a modulated, low but detectable,5′-to-3′ exonuclease activity compared to wild type Tne DNA polymerase.M13/pUC Forward 23-Base Sequencing Primer™, obtainable from LTI,Rockville, Md., was labeled at the 5′ end with [P³²] ATP and T4 kinase,also obtainable from LTI, Rockville, Md., as described by themanufacturer. The reaction mixtures contained 20 units of eitherwildtype or mutant Tne DNA polymerase, 0.25 pmol of labeled primer, 20mM tricine, pH 8.7, 85 mM potassium acetate, 1.2 mM magnesium acetate,and 8% glycerol. Incubation was carried out at 70° C. At various timepoints, 10 μl aliquots were removed to 5 μl cycle sequencing stopsolution and were resolved in a 6% polyacrylamide sequencing gelfollowed by andoradiography. While the wildtype polymerase degraded theprimer in 5 to 15 minutes, it took the mutant polymerase more than 60minutes for the same amount of degradation of the primer.

EXAMPLE 12

[0213] Purification of the Mutant Polymerases

[0214] The purification of the mutant polymerases was done essentiallyas described Example 6, supra, with minor modifications. Specifically, 5to 10 grams of cells expressing cloned mutant Tne DNA polymerase werelysed by sonication with a Heat Systems Ultrasonic, Inc. Model 375machine in a sonication buffer comprising 50 mM Tris-HCl (pH 7.4); 8%glycerol; 5 mM 2-mercaptoethanol, 10 mM NaCl, 1 mM EDTA, and 0.5 mMPMSF. The sonication sample was heated at 75° C. for 15 minutes.Following heat treatment, 200 mM NaCl and 0.4% PEI was added to removenucleic acids. The extract was centrifuged for clarification. Ammoniumsulfate was added to 48%, the pellet was resuspended in a column bufferconsisting of 25 mM Tris-HCl (pH 7.4); 8% glycerol; 0.5% EDTA; 5 mM2-mercaptoethanol; 10 mM KCl and loaded on a heparin agarose (LTI)column. The column was washed with 10 column volumes using the loadingbuffer and eluted with a 10 column volume buffer gradient from 10 mM to1 M KCl. Fractions containing polymerase activity were pooled anddialyzed in column buffer as above with the pH adjusted to 7.8. Thedialyzed pool of fractions were loaded onto a MonoQ (Pharmacia) column.The column was washed and eluted as described above for the heparincolumn. The active fractions are pooled and a unit assay was performed.

[0215] The unit assay reaction mixture contained 25 mM TAPS (pH 9.3), 2mM MgCl₂, 50 mM KCl, 1 mM DTT, 0.2 mM dNTPs, 500 μg/ml DNAse I treatedsalmon sperm DNA, 21 mCi/ml [αP³²] dCTP and various amounts ofpolymerase in a final volume of 50 μl. After 10 minutes incubation at70° C., 10 μl of 0.5 M EDTA was added to the tube. TCA perceptiblecounts were measured in GF/C filters using 40 μl of the reactionmixture.

EXAMPLE 13

[0216] Generation of 5′-to-3′ exonuclease mutant of full length Tne DNApolymerase

[0217] 1. Identification of Two Amino Acids Responsible for 5′-to-3′Exonuclease Activity

[0218] Tne DNA polymerase contains three enzymatic activities similar toE. coli DNA polymerase I:5′-to-3′DNA polymerase activity, 3′-to-5′exonuclease activity and 5′-to-3′ exonuclease activity. This example isdirected to the elimination of the 5′-to-3′ exonuclease activity in fulllength Tne DNA polymerase. Gutman and Minton (Nucleic Acids Res. 1993,21, 4406-4407) identified six (A-F) conserved 5′-to-3′ exonucleasedomains containing a total of 10 carboxylates in various DNA polymerasesin the polI family. Seven out of 10 carboxylates (in domains A, D and E)have been implicated to be involved in divalent metal ions binding asjudged from the crystal structure (Kim et al. Nature, 1995, 376,612-616) of Taq DNA polymerase. However, there was no cleardemonstration that these carboxylates are actually involved 5′-to-3′exonuclease activity. In order to find out the biochemicalcharacteristics of some of these carboxylates, two of the aspartic acidsin domains A and E were chosen for mutagenesis. The following asparticacids in these two domains were identified: Tne DNA polymerase: 5 FLFD⁸GT 10 (domain A) (SEQ ID NO:24) Taq DNA polymerase: 15 LLVD ¹⁸GH 20(SEQ ID NO:25) and Tne DNA polymerase: 132 SLITGD ¹³⁷KDML141 (domain E)(SEQ ID NO:26) Taq DNA polymerase: 137 RILTAD ¹⁴²KDLY146 (SEQ ID NO:27)

[0219] 2. Isolation of Single Stranded DNA for Mutagenesis

[0220] Single stranded DNA was isolated from pSportTne (see infra).pSportTne was introduced into DH5αF′IQ (LTI, Rockville, Md.) bytransformation. A single colony was grown in 2 ml Circle Grow (Bio 101,CA) medium with ampicillin at 37° C. for 16 hrs. A 10 ml fresh media wasinoculated with 0.1 ml of the culture and grown at 37° C. until the A590reached approximately 0.5. At that time, 0.1 ml of M13KO7 helper phage(1×10¹¹ pfu/ml, LTI) was added to the culture. The infected culture wasgrown for 75 min. Kanamycin was then added at 50 μg/ml, and the culturewas grown overnight (16 hrs.). The culture was spun down. 9 ml of thesupernatant was treated with 50 μg each of RNaseA and DNaseI in thepresence of 10 mM MgCl₂ for 30 min. at room temperature. To thismixture, 0.25 volume of a cocktail of 3M ammonium acetate plus 20%polyethylene glycol was added and incubated for 20 min. on ice toprecipitate phage. The phage was recovered by centrifugation. The phagepellet was dissolved in 200 μl of TE (10 mM Tris-HCl (pH 8) and 1 mMEDTA). The phage solution was extracted twice with equal volume ofbuffer saturated phenol (LTI, Rockville, Md.), twice with equal volumeof phenol:chloroform:isoamyl alcohol mixture (25:24:1, LTI, Rockville,Md.) and finally, twice with chloroform:isoamyl alcohol (24:1). To theaqueous layer, 0.1 volume of 7.5 M ammonium acetate and 2.5 volume ofethanol were added and incubated for 15 min. at room temperature toprecipitate single stranded DNA. The DNA was recovered by centrifugationand suspended in 200 μl TE.

[0221] 3. Mutagenesis of D⁸ and D¹³⁷

[0222] Two oligos were designed to mutagenize D⁸ and D¹³⁷ to alanine.The oligos are: 5′ GTAGGCCAGGGCTGTGCCGGCAAAGAGAAATAGTC 3′(D8A) (SEQ IDNO:28) and 5′GAAGCATATCCTTGGCGCCGGTTAT TATGAAAATC 3′ (D137A) (SEQ IDNO:29). In the D8A oligo a NgoAIV (bold underlined) and in the oligoD137A a KasI (bold underlined) site was created for easy identificationof clones following mutagenesis. 200 pmol of each oligo was kinasedaccording to the Muta-gene protocol (Bio-Rad, CA) using 5 units of T4Kinase (LTI, Rockville, Md.). 200 ng of single stranded DNA was annealedwith 2 pmol of oligo according to the Muta-gene protocol. The reactionvolume was 10 μl. Following the annealing step, complementary DNAsynthesis and ligation was carried out using 5 units of wildtype T7 DNApolymerase (USB, Ohio) and 0.5 unit T4 ligase (LTI). 1 μl of thereaction was used to transform a MutS E. coli (obtainable from Dr. PaulModrich at the Duke University, N.C.) and selected in agar platescontaining ampicillin. A control annealing and synthesis reaction wascarried out without addition of any oligo to determine the background.There were 50-60 fold more colonies in the transformation plates withthe oligos than without any oligo. Six colonies from each mutagenicoligo directed synthesis were grown and checked for respectiverestriction site (NgoAIV or KasI). For D8A (NgoAIV), 4 out of 6generated two fragments (3 kb and 4.1 kb). Since pSportTne has an NgoAIVsite near the f1 intergenic region, the new NgoAIV site within the TneDNA polymerase produced the expected fragments. The plasmid wasdesignated as pSportTneNgoAIV. For D137A (KasI), 5 out of 6 clonesproduced two expected fragments of 1.1 kb and 6 kb in size. SincepSportTne has another KasI site, the newly created KasI site generatedthese two expected fragments. The plasmid was designated aspSportTneKasI. Both D8A and D137A mutations were confirmed by DNAsequencing.

[0223] 4. Reconstruction of the Mutant Polymerase into Expression Vector

[0224] During the course of expression of Tne DNA polymerase or mutantTne DNA polymerase, a variety of clones were constructed. One such clonewas designated as pTTQ Tne SeqS 1. This plasmid was constructed asfollows: first, similar to above mutagenesis technique glycine 195 waschanged to an aspartic acid in pSportTne. A mutation in thecorresponding amino acid in E. coli DNA polymerasel (polA214, domain F)was found to have lost the 5′-to-3′ exonuclease activity (Gutman andMinton, see above). An SspI site was created in the mutant polymerase.Second, a 650 bp SstI-SphI fragment containing the G195D mutation wassubcloned in pUCTne35FY (see infra) to replace the wild type fragment.This plasmid was called pUCTne3022. Finally, the entire mutant Tne DNApolymerase was subcloned from pUCTne3022 into pTTQ18 as SstI-HindIIIfragment to generate pTTQTneSeqS1. To introduce the mutation D8A orD137A in this expression vector, the 650 bp SstI-SphI was replaced withthe same SstI-SphI fragment from pSportTneNgoAIV or pSportTneKasI. Theplasmids were designated as pTTQTneNgo(D8A) and pTTQTneKas(D137A),respectively.

[0225] 5. Confirmation of the Mutations by DNA Sequencing

[0226] DNA sequencing of both mutant polymerases confirmed the presenceof the restriction site NgoAIV as well as the mutation D8A; and KasIsite as well as the mutation D137A. Also confirmed by DNA sequencing wasthe presence of the mutation D323A and the Eco47III restriction site inthe 3′-to-5′ exonuclease region. In addition, confirmed by DNAsequencing was the F730Y mutation and the HpaI restriction site in theO-helix region of the mutant Tne DNA polymerase.

[0227] 6. 5′-to-3′ exonuclease Activity of the Mutant Tne DNAPolymerases

[0228] The full length mutant DNA polymerase was purified as describedabove. The 5′-to-3′exonuclease activity was determined as described inthe LTI catalog. Briefly, 1 pmol of labeled (³²p) HaeIII digested λ DNA(LTI) was used for the assay. The buffer composition is: 25 mM Tris-HCl(pH 8.3), 5 MM MgCl₂, 50 mM NaCl, 0.01% gelatin. The reaction wasinitiated by the addition of 0, 2, 4, 6 and 10 units of either wild typeor mutant Tne DNA polymerase in a 50 μl reaction. The reaction mix wasincubated for 1 hr at 72° C. A 10 μl aliquot was subjected toPEI-cellulose thin layer chromatography and the label released wasquantitated by liquid scintillation. In this assay, both D8A and D137Amutants showed less than 0.01% label release compared to the wild typeTne DNA polymerase. The result demonstrates that in both D8A and D137Amutants the 5′-to-3′ exonuclease activity has been considerablydiminished. Thus, it has been confirmed that these two aspartates areinvolved with the 5′-to-3′ exonuclease activity.

EXAMPLE 14

[0229] Generation of Double Mutants, R722K/F730Y, R722Q/F730Y,R722H/F730Y and R722N/F730Y of Tne DNA polymerase

[0230] For all mutations, the PCR method was used. A common 5′-oligo,CAC CAG ACG GGT ACC GCC ACT GGC AGG TTG (SEQ ID NO:30), was used. Thisoligo contains a KpnI site (shown above in bold italics). The templateused for PCR was pTTQTneSeqS1 (Example 13) which already contains theF730Y mutation in the Tne polymerase gene. For the R722K/F730Y mutation,the oligo used was TAT AGA GTA GTT AAC CAT CTT TCC AAC CCG TTT CAT TTCTTC GAA CAC (SEQ ID NO:31). For the R722Q/F730Y mutation, the oligo usedwas TAT AGA GTA GTT AAC CAT CTT TCC AAC CCG TTG CAT TTC TTC GAA CAC (SEQID NO:32). For the R722N/F730Y mutation, the oligo used was TAT AGA GTAGTT AAC CAT CTT TCC AAC CCG GTT CAT TTC TTC GAA CAC (SEQ ID NO:33) andfor the R722H/F730Y the oligo used was TAT AGA GTA GTT AAC CAT CTT TCCAAC CCG ATG CAT TTC TTC GAA CAC (SEQ ID NO:34). Each of these oligoscontains a HpaI site (bold italics). The underlined codons were themutated codons for arginine at the position 722 for respective aminoacids. The PCR generated a 318 bp product containing a KpnI and a HpaIsite. The PCR products were digested with KpnI and HpaI and cloned intopUC-TneFY digested with KpnI and HpaI to replace the original fragmentto generate pUC19TneFY-R722K, pUC19TneFY-R722Q, pUC19TneFY-R722H andpUC19TneFY-R722N. Finally, the KpnI-HindIII fragment (˜800 bp) ofpTTQTneKasI(D137A) was replaced by the ˜800 bp KpnI-HindIII fragmentfrom these plasmids to generate pTne11 (R722K/F730Y), pTne10(R722Q/F730Y), pTne13 (R722H/F730Y) and pTne9 (R722N/F739Y),respectively. The mutations were confirmed by DNA sequencing.

EXAMPLE 15

[0231] Generation of Tne DNA Polymerase Mutants F730A and F730S

[0232] F730A was constructed using PCR. The forward oligo was AAG ATGGTT AAC GCG TCT ATA ATA TAC GG (SEQ ID NO:35) which contains a HpaI siteand a MluI site (bold italics). The reverse oligo was CAA GAG GCA CAGAGA GTT TCA CC (SEQ ID NO:36) which anneals downstream of SpeI presentin the Tne polymerase gene. The template used for PCR was pTTQTne KasI(D137A). The 482 bp PCR product was digested with HpaI and SpeI andcloned into pUC-TneFY thereby replacing the amino acid tyrosine atposition 730 with alanine. This construct was called pUC-Tne FA.

[0233] F730S was constructed by site directed mutagenesis. The oligo wasGTA TAT TAT AGA GGA GTT AAC CAT CTT TCC (SEQ ID NO:37) where a HpaI sitewas created (bold italics). The single stranded DNA used was isolatedfrom pSport-Tne that contains the double mutation D137A and D323A. Thisconstruct was designated pTne 47. The Tne polymerase gene was thencloned as an SstI and HindIII fragment into the plasmid pUC19 and theresulting clone was designated pTne101.

EXAMPLE 16

[0234] Generation of Tne DNA Polymerase with a HpaI Site in Front of theAmino Acid Phenylalanine at Position 730.

[0235] A construct of Tne polymerase was made using PCR where a HpaIrestriction enzyme site was introduced into the gene in front of theamino acid phenylalanine at position 730. The forward oligonucleotidewas AAG ATG GTT AAC TTC TCT ATA ATA TAC GG (SEQ ID NO:38) which containsa HpaI site (shown above in bold italics) and the reverse oligo was thesame as in Example 15 above. The template used for PCR was pTne33 whichcontains the Tne polymerase gene with D137A and D323A mutations clonedin pUC19. The 482bp PCR product was digested with HpaI and Spel and wasused to replace the corresponding fragment in pTne101 (see example 15).The construct was sequenced to verify that the amino acid at position730 was indeed phenylalanine and the plasmid was numbered pTne106.

Example 17

[0236] Generation of Double Mutants R722Y/F730A and R722L/F730A of theTne DNA Polymerase

[0237] For both the mutations PCR method was used. The common 5′ oligowas the same as in Example 14. For R722Y/F730A mutation the oligo usedwas TAT AGA GTA GTT AAC CAT CTT TCC AAC CCG GTA CAT GTC TTC GTT CAC (SEQID NO:39). For R722L/F730A mutation the oligo used was TAT AGA GTA GTTAAC CAT CTT TCC AAC CCG CAA CAT GT C TTC GTT CAC (SEQ ID NO:40). Each ofthese oligos contain a HpaI site (shown above in bold italics). Theunderlined codons were the mutated codons for arginine at the position722 for respective amino acids. An AflIII site was also created (shownabove in bold italics next to the underlined codon) in order to confirmthe mutation. The PCR generated a 318 bp product containing a KpnI and aHpaI site. The PCR products were digested with KpnI and HpaI and clonedinto pUC-TneFA (see example 15). The constructs were named as pUCTneYAand pUCTneLA.

EXAMPLE 18

[0238] Generation of Tne DNA Polymerase Mutants R722Y and R722L.

[0239] The plasmid pTne 106 (see example 16) was digested with HpaI andKpnI and the 318 bp fragment was replaced with the correspondingfragment from pUCTneYA or pUCTneLA (see Example 17) to generate themutants R722Y or R722L. In these constructs the amino acid at position730 is the same as wild type Tne (phenylalanine). The constructs weresequenced to confirm the R722Y and the R722L mutations. The Tne DNApolymerase gene was then cloned as a SstI/HindIII fragment into theplasmid pSport1.

EXAMPLE 19

[0240] Generation of Tne DNA Polymerase Mutants R722K, R722Q and R722H.

[0241] The construct pTne 106 (see example 16) was digested with HpaIand KpnI and the 318 bp fragment was replaced with the correspondingfragment from the construct pUC19TneFY-R722K, pUC19TneFY-R722H or pTne10(see Example 14), to generate the mutants R722K, R722H and R722Q. Theconstructs were sequenced to confirm the mutations. The Tne DNApolymerase gene was then subcloned into the vector pSport1 as aSstI/HindIII fragment.

EXAMPLE 20

[0242] Purification of the mutant Tne DNA Polymerases

[0243] The purification of the mutants of Tne DNA polymerase was carriedout based on the method described above with minor modifications. Two tothree grams of cells expressing cloned mutant Tne DNA polymerase wereresuspended in 15-20 ml of sonication buffer (50 mM Tris-HCl, pH 8.0,10% glycerol, 5 mM 2-mercaptoethanol, 50 mM NaCl, 1 mM BDTA, and 0.5 mMPMSF and sonicated with a 550 Sonic Dismembrator (Fisher Scientific).The sonicated sample was heated at 82° C. for 20 min and then cooled inice-water for 5 min. In the sample, 20 mM NaCl and 0.2% PEI were addedand centrifuged at 13,000 rmp for 10 min. Ammonium sulfate (305 g/L) wasadded to the supernatant. The pellet was collected by centrifugation andresuspended in 4 ml of MonoQ column buffer (50 mM Tris-HCl, pH 8.0, 10%glycerol, 5 mM 2-mercaptoethanol, 50 mM NaCl, and 1 mM EDTA). The samplewas dialyzed against one litter of MonoQ buffer overnight. Following thecentrifugation at 13,000 rpm to remove any insoluble materials, thesample was loaded onto a MonoQ column (HR5/5, Pharmacia). The column waswashed with MonoQ column buffer to baseline of OD₂₈₀ and then elutedwith a linear gradient of 50-300 mM NaCl in 20 ml MonoQ column buffer.The fractions were analyzed by 8% SDS-PAGE and the Tne DNA polymeraseactivity determined as described earlier. The fractions containingactive and pure Tne DNA polymerase were pooled.

EXAMPLE 21

[0244] Generation of Taq DNA Polymerase Mutants R659K, R659H and R659Y

[0245] A 2.5 kb portion of the gene encoding Taq DNA polymerase (FIG. 5)was cloned as a Hind III-Xba I fragment into M13mp19. Site directedmutagenesis was performed using the BioRad mutagene kit (BioRadCalifornia) using the following oligonucleotides:

[0246] CTTGGCCGCCCGATGCATCAGGGGGTC (SEQ ID NO:41) for the R659H mutationwhere an NsiI site was created (see bold italics);

[0247] CTTGGCCGCCCGCTTCATGAGGGGGTCCAC (SEQ ID NO:42) for the R659Kmutation where a BspHI site was created (see bold italics); and

[0248] CTTGGCCGCCCTGTACATCAGGGGGTC (SEQ ID NO:43) for the R659Y mutationwhere a BsrGI site was created (see bold italics).

[0249] For each mutation, six clones were screened by analyzing theM13RF DNA for the expected restriction sites. Mutations were confirmedby DNA sequencing. DNA shown to contain the mutation by the presence ofthe expected restriction site was digested with NgoAIV and Xba I and theapproximately 1600 base pair fragment was used to replace correspondingfragment in the wildtype Taq DNA polymerase gene. These constructs weremade in a plasmid containing Taq polymerase gene under the control ofTac promoter (pTTQ Taq) to generate pTTQ Taq (R659K), PTTQ Taq (R659H)and pTTQ Taq (R659Y). These plasmids were transformed into E. Coli DH10B(LTI).

EXAMPLE 22

[0250] Construction of Tne Polymerase Mutants Containing F730S and F730T

[0251] Single stranded DNA was isolated from pSportTne (Tne35)containing D137A and D323A mutations as described in the section 2 ofexample 13. These D137 and D323A mutations rendered Tne DNA polymerasedevoid of 5′-exonuclease and 3′-to-5′-exonuclease activities,respectively. Thus, Tne 35 is devoid of both exonuclease activities. Thesite-directed mutagenesis was done following the protocol decribed insection 3 of Example 13. The oligos used were 5′ GTA TAT 5′GTA TAT TATAGA GGA GTT AAC CAT CTT TCC 3′ (SEQ ID NO:37) for F730S and 5′GTA TATTAT AGA GGT GTT AAC CAT CTT TCC 3′ (SEQ ID NO:44)

[0252] for F730T. Each of these two oligos contain a diagonistic HpaIsite for screening of mutants in the MutS strain. The mutant plasmidswere transferred to DH10B strains. The mutations were finally confirmedby DNA sequencing. The mutant polymerases were purified by the procedureas described in Example 20.

EXAMPLE 23

[0253] Determination of the Activity of Non-templated One Base Additionfor Tne and Taq DNA Polymerase by Primer Extension Assay

[0254] The following 34-mer primer was ³²P labeled at the 5′ end with[γ-³²P] ATP and T4 polynucleotide kinase by standard protocol (MolecularCloning, A Laboratory Manual, Cold Spring Harbor, N.Y.):5′-GGGAGACCGGAATTCTCCTTCATTAATTCCTATA-3′ (SEQ ID NO:45)

[0255] The unincorporated ATP was removed by a BioRad P6 column(1.0 ml).The labeled primer was annealed to the following homogenous (purified)48-mer template: 5′-TGGAGACCCTGGAACTATAGGAATTAATGAA (SEQ ID NO:46).GGAGAATTCCGGTCTCCC-3′

[0256] Wildtype or mutant DNA polymerases (0.125-1.0 unit) wereincubated at 72° C. for 2 min in 20 mM Tris-HCl (pH8.3), 1.5 mM MgCl₂,50 mM KCl, 1.0 mMDTT, 200 uM of dCTP, dGTP, TTP, dATP, and 0.02 pmol ofthe annealed primer-template. After addition of sequencing stop bufferand heated at 90° C. for 2 min, the mixture was loaded onto 10%polyacrylamide-7 M urea. Following the electrophoresis, the gel wasdried and the reaction products were analyzed by autoradiography. Thenon-templated one base addition products shown in FIG. 6 were quantifiedby a PhosphorImager (Molecular Dynamics). % of N + 1 Tne DNA polymerases 1 D137A 18.5  2 D137A D323A 78.5  3 D137A D323A R722K 0.7  4 D137AD323A R722Y 0.7  5 D137A D323A R722L 5.7  6 D137A D323A R722H 1.2  7D137A D323A R722Q 1.4  8 D137A D323A F730Y 61.3  9 D137A D323A R722KF730Y 6.8 10 D137A D323A R722H F730Y 2.1 11 D137A D323A R722Q F730Y 6.112 D137A D323A R722N F730Y 15.9 13 D137A D323A F730S 8.3 14 D137A D323AF730T 24.2 Taq DNA Polymerases  1 W.T. 37  2 R659K 1.4  3 R659Y 0.9  4R659H 0.5  5 F667Y 39.1

EXAMPLE 24

[0257] Comparison of DNA Synthesis by Taq and Tne

[0258] To examine its propensity to add a nontemplated nucleotides tothe 3′ termini of PCR products, Tne DNA polymerase (5′exo⁻, 3′exo−) wascompared side-by-side with Taq DNA polymerase in amplifications of shorttandem repeats at 23 different marker loci (see Table 1). Reactionscomprising 20 mM TRIS-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 200 mM eachdNTP, 200 nM [³²P] α-dATP, 200 nM each of the upper and lower primers,25 ng of human DNA, 0.1% nonionic detergent and 1 unit of DNA polymerase(in a volume of 25 ml) were assembled on ice. Published sequences forupper and lower primers for each locus, as shown in Table 1, were usedfor all amplifications.

[0259] Reactions were loaded into a Perkin Elmer model 9600 thermocyclerpreheated to 94° C. and PCR was done using standard cycling conditions(1 minute pre-denaturation at 94° C.; 30 cycles of 30 seconds at 94° C.,30 seconds at 55° C., and 1 minute at 72° C.; 1 minute post-extension at72° C.; overnight soak at 4° C.). A portion of each reaction was mixedwith an equal volume of 95% formamide containing dyes to indicate theprogress of electrophoresis. Samples were heated to 90° C. for 2 min,and 5 ml of each was loaded on a 6% denaturing polyacrylamide gel.Sequencing ladders were loaded to provide size markers, andelectrophoresis was performed at 70 watts. After electrophoresis the gelwas transferred to filter paper and dried. Autoradiography andphosphoimage analysis was performed to visualize the PCR products andestimate the percentage of product which contained the added nucleotideby direct comparison of bands produced by each enzyme.

[0260] Examples of the side-by-side comparisons of amplificationproducts produced by Taq DNA polymerase and Tne DNA polymerase are shownin FIG. 7 for the CD4 locus and in FIG. 8 for the D20S27 locus. At bothof these loci, a significant portion of the Taq PCR product contained anextra non-templated nucleotide (n+1), while Tne polymerase demonstratedno apparent non-templated nucleotide incorporation for either the CD4locus (FIG. 7) or the D20S27 locus (FIG. 8). Complete results for the 23marker loci examined are summarized in Table 2. In PCRs using Taq DNApolymerase, a portion of the amplification product contained an extranon-templated nucleotide (n+1) at every locus examined. In PCRs usingTne DNA polymerase, however, no detectable portion of the product at anyof the loci examined contained an additional non-templated nucleotide.These results indicate that Tne DNA polymerase, in contrast to Taq DNApolymerase, is substantially reduced in the ability to add anontemplated 3′ terminal nucleotide to the growing strand. Since the TneDNA polymerase used in these amplifications was a 3′exo− mutant (i.e.,it was substantially reduced in 3′ exonuclease activity), these resultsare consistent with the notion that the Tne polymerase was unable to addthe extra nucleotide to the product rather than adding the nucleotideand then removing it via a 3′ exonuclease activity. TABLE Primers Usedin Example 24. Locus Upper Primer (SEQ ID NO:) Lower Primer (SEQ ID NO:)Reference D13S71 GTATTTTGGTATGCTTGTGC (47) CTATTTTGGAATATATGTGCCT (48)Nucl. Acids Res. 18:4638 (1990) D1S103 ACGAACATTTCTACAAGTTAC (49)TTTCAGAGAAACTGACCTGT (50) Nucl. Acids Res. 18:2199 (1990) D15S87GATAAATGCCAAACATGTTGT (51) TGCTCTCAGGATTTCCTCCA (52) Nucl. Acids Res.18:4640 (1990) D2S136 AGCTTGAGACCTCTGTGTCC (53) ATTCAGAAGAAACAGTGATGGT(54) Nature Genet. 7:246-339 (1994) CD4 TTGGAGTCGCAAGCTGAACTAGC (55)GCCTGAGTGACAGAGTGAGAACC (56) Nucl. Acids Res. 19:4791 (1991) PLA2ACCCACTAGGTTGTAAGCTCCATGA(57) TACTATGTGCCAGGCTCTGTCCTA (58) Nucl. AcidsRes. 18:7468 (1990) D19S49 ACTCATGAAGGTGACAGTTC (59)GTGTTGTTGACCTATTGCAT (60) Nucl. Acids Res. 18:1927 (1990) D4S175ATCTCTGTTCGCTCCCTGTT (61) CTTATTGGCCTTGAAGGTAG (62) Genomics 14:209-219(1992) APOC2AC AGCCCGTGTTGGAACCATGACTG (63) TACATAGCGAGACTCCATCTCCC (64)Hum. Genet. 83:245-251 1989 D20S27 TTTATGCGAGCGTATGGATA (65)CACCACCATTGATCTGGAAG (66) Nucl. Acids Res. 18:2202 (1990) D15S127CCAACCACACTGGGAA (67) AACAGTTGCGCACGGT (68) Nature Genet. 7:246-339(1994) D4S398 CATGAAATGCTGACTGGGTA (69) TCAATTTATGTGCAGCCAAT (70) NatureGenet. 7:246-339 (1994) APOC2 CATAGCGAGACTCCATCTCC (71)GGGAGAGGGCAAAGATCGAT (72) Am. J. Hum. Genet. 44:388-396 (1989) D10S89AACACTAGTGACATTATTTTCA (73) AGCTAGGCCTGAAGGCTTCT (74) Nuci. Acids Res.18:4637 (1990) VWA CCCTAGTGGATGATAAGAATAATC (75)GGACAGATGATAAATACATAGGATGGATGG (76) Hum. Molec. Genet. 1:287 (1992)D16S401 TTCTCTTACAACACTGCCCC (77) ATTTGGATGGCTTGACAGAG (78) NatureGenet. 7:246-339 (1994) D7S440 ACATTGTAAGACTTTCCCAAT (79)AGAGCATGCACCCTGAATTG (80) Nucl. Acids Res. 18:4039 1990 D4S174AAGAACCATGCGATACGACT (81) CATTCCTAGATGGGTAAAGC (82) Nucl. Acids Res.18:4636 (1990) D16S520 GCTTAGTCATACGAGCGG (83) TCCACAGCCATGTAAACC (84)Nature Genet. 7:246-339 (1994) D16S511 GCCCGGAGCAAGTTCA (85)CAGCCCAAAGCCAGATTA (86) Nature Genet. 7:246-339 (1994) D21S11ATATGTGAGTCAATTCCCCAAG (87) TGTATTAGTCAATGTTCTCCAG (88) Hum. Molec.Genet. 1:67 (1992) TH01 CAGCTGCCCTAGTCAGCAC (89) GCTTCCGAGTGCAGGTCACA(90) Nucl. Acids Res. 19:3753 (1991) ACTBP2 ATTCTGGGCGCACAAGAGTGA (91)ACATCTCCCCTACCGCTATA (92) Nucl. Acids Res. 20:1432 (1992)

[0261] TABLE 2 Non-templated 3′ Terminal Nucleotide Addition by Taq andTne DNA Polymerases at 23 Microsatellite DNA Loci. Locus Repeat Type Taq(% n + 1) Tne (% n + 1) D13S71 dinucleotide 100 0 D1S103 dinucleotide75-100 0 D15S87 dinucleotide 30-50  0 D2S136 dinucleotide 90-100 0HUMCD4 pentanucleotide 50 0 HUMPLA2A trinucleotide 25 0 D19S49dinucleotide 75 0 D4S175 dinucleotide 75 0 APOC2AC dinucleotide 50 0D20S27 dinucleotide 50 0 D15S127 dinucleotide 100 0 D4S398 dinucleotide50 0 APOC2 dinucleotide 50 0 D10S89 dinucleotide 75-100 0 HUMVWAtetranucleotide 90 0 D16S401 dinucleotide 100 0 D7S440 dinucleotide 90 0D4S174 dinucleotide 75 0 D16S520 dinucleotide 100 0 D16S511 dinucleotide100 0 HUMD21S11 tetranucleotide 100 0 HUMTHO1 tetranucleotide 75 0HUMACTBP2 tetranucleotide 25 0

EXAMPLE 25

[0262] Comparison of DNA Synthesis by Tne and Other Thermostable Enzymes

[0263] To further evaluate the differences in the propensities of Tneand other thermostable DNA polymerases to add non-templated 3′ terminalnucleotides to PCR products, side-by-side amplifications were performedusing a single marker locus D1S103 and a variety of thermostableenzymes, including 3′ exonuclease deficient (3′exo−) enzymes, and 3′exonuclease competent (3′exo+) enzymes. PCR amplifications,electrophoresis and analysis were performed as described for Example 24,using 200 nM of D1S103-specific upper and lower primers.

[0264] Results for the amplifications using 3′exo− DNA polymerases areshown in Table 3. With the exception of Tne(3′exo−), all of the 3′exo−DNA polymerases examined exhibited a propensity to add a non-templated3′ terminal nucleotide (n+1) to the PCR product. For Taq and Tbr DNApolymerases, up to 100% of the PCR products contained an additionalnon-templated 3′ terminal nucleotide, while Vent, Deep Vent, and Dtok3′exo− mutants polymerases added this non-templated nucleotide to25-100% of the PCR products. In contrast, the 3′exo− mutant of Tne DNApolymerase was substantially reduced in the ability to add anontemplated 3′ terminal nucleotide to the DNA molecule; none of the PCRproducts from reactions using Tne(3′exo−) had an additionalnon-templated nucleotide at their 3′termini.

[0265] Results from amplifications using 3′exo+ DNA polymerases areshown in Table 4. Five polymerases were examined as well as twocommercially available enzyme mixes (mixtures of a primary 3′exo−polymerase and a secondary 3′exo+polymerase). At this locus, the 3′exo+DNA polymerases (Tne, Tma, Pfu, Pwo and 9°North) yielded product whichdid not contain an extra non-templated nucleotide. The enzyme mixtures(Elongase and Expand HiFi) yielded a mixture of products with andwithout an additional non-templated nucleotide. Together, these resultsindicate that Tne polymerases, whether 3′exo− or 3′exo+, aresubstantially reduced in the ability to add a nontemplated 3′terminalnucleotide to the DNA molecule. Moreover, of the preferred 3′exo−polymerases, only Tne(3′exo−) was substantially reduced in thisactivity, indicating its favorableness in PCR applications wherenon-templated nucleotide addition to the amplification product isundesirable. TABLE 3 Non-templated 3′ Terminal Nucleotide Addition by3′exo− DNA Polymerases. Enzyme n Sized Fragment n + 1 Sized FragmentTne(3′exo−) + − Taq − + Vent(3′exo−) + + Deep Vent(3′exo−) + +Dtok(3′exo−) + + ThermolaseTbr − +

[0266] TABLE 4 Non-templated 3′ Terminal Nucleotide Addition by 3′exo+DNA Polymerases. Enzyme n Sized Fragment n + 1 Sized FragmentTne(3′exo+) + − UlTma + − Pfu + − Pwo + − 9° North + − Elongase + +Expand HiFi + +

EXAMPLE 26

[0267] Comparison of DNA Synthesis by Tne Mutants

[0268] To examine the utility of Tne DNA polymerase and various mutantsthereof in amplification of microsatellite DNA sequences, theexperiments described in Example 25 were repeated with 11 different TneDNA polymerase mutants. Of these mutants, 3 were 5′exo+, while theremainder were 5′exo− either due to N-terminal deletions of the protein,or to point mutations in the 5′exonuclease domain of the polymerase.

[0269] As shown in Table 5, use of the 5′exo− Tne mutants resulted inproductive amplifications, yielding PCR products with no non-templated3′terminal nucleotide additions. Results were identical for all sevenTne(3′exo−/5′exo−) polymerase mutants, as well as for the singleTne(3′exo+/5′exo−) mutant tested. Results with 5′exo+ Tne mutants wereinconclusive under the conditions tested.

[0270] These results indicate that the mutants of Tne DNA polymerasetested in the present studies are substantially reduced in the abilityto add nontemplated 3′ terminal nucleotides to the growing strand,particularly a DNA template comprising a microsatellite DNA sequence oran STR. TABLE 5 Non-templated 3′ Terminal Nucleotide Addition by Tne DNAPolymerase Mutants 5′exo 3′exo n Sized n + 1 Sized Enzyme ActivityActivity Fragment Fragment Tne N′Δ219, D323A − − + − Tne N′Δ283, D323A −− + − Tne N′Δ192, D323A − − + − Tne D137A, D323A − − + − Tne D8A, D323A− − + − Tne G195D, D323A − − + − Tne G37D, D323A − − + − Tne N′Δ283− + + −

EXAMPLE 27

[0271] Fluorescent Analysis of DNA Synthesis by Tne and Taq DNAPolymerases

[0272] In an alternative analysis approach, the propensities of Taq DNApolymerase and Tne DNA polymerase to add non-templated nucleotides tothe PCR products were compared using fluorescent detection. Thepolymerases were compared in side-by-side amplifications utilizing acommonly used commercially available marker panel (ABI Prism LinkageMapping Set Panel 21), examining ten different loci. Reaction mixtures(15 ml) containing 1.5 mM MgCl₂, 250 mM of each deoxynucleosidetriphosphate, 333 nM of each primer, 50 ng of human DNA and 0.6 units ofTaq or Tne DNA polymerase were assembled on ice. Reactions were loadedinto a Perkin Elmer model 9600 thermocycler preheated to 95° C., and PCRwas performed using recommended cycling conditions (5 minutespre-denaturation at 95° C.; 10 cycles of 15 seconds at 95° C., 15seconds at 55° C., and 60 seconds at 72° C.; and 20 cycles of 15 secondsat 89° C., 15 seconds at 55° C., and 60 seconds at 72° C.). Two sets ofextension reactions were conducted for each locus, one with a 10 minutepost-extension incubation at 72° C. followed by an overnight soak andstorage at 4° C. (conditions which favor nontemplated 3′ nucleotideaddition), the other with no post-extension incubation followed byimmediate storage at −20° C. (conditions which inhibit nontemplated 3′nucleotide addition). A portion of each reaction was diluted, mixed withloading cocktail, heat denatured and loaded on an 8% polyacrylamidesequencing gel. The ABI 373 Stretch Automated Sequencer was run for 5-6hours at 15 W in order to obtain single base resolution, and data wereanalyzed using GeneScan software. Areas of the peaks recognized by thesoftware were used to estimate the percentage of nontemplated 3′nucleotide addition (“n+1”) for each locus by the two polymerases underthe two different extension conditions. The total area under the allelicpeaks was used to compare the yields of specific PCR product obtained inTne and Taq amplifications, and yields produced by Tne polymerase wereexpressed for each locus as a percentage of those produced by Taqpolymerase. Table 6 summarizes the results obtained. TABLE 6 Comparisonof DNA Amplification by Taq and Tne DNA Polymerases by FluorescentDetection Tne yield expected cycling Taq Tne (% locus color sizeconditions pattern pattern Taq) D16S40 blue 107-145 no final ext 0% n +1 100% n  89% 5 10′ final 94% n + 1 100% n 178% ext D15S12 green 114-148no final ext 53% n + 1 100% n 133% 7 10′ final 100% 100% n 142% ext n +1 D16S52 yellow 144-160 no final ext 40% n + 1  98% n 275% 0 10′ final100% 100% n 252% ext n + 1 D16S51 green 182-222 no final ext 62% n + 1100% n  51% 1 10′ final 100% 100% n 160% ext n + 1 D16S41 blue 215-235no final ext 0% n + 1 100% n 218% 1 10′ final 64% n + 1 100% n 257% ext67% n + 1  95% n 305% 10′ final ext D15S13 yellow 237-275 no final ext0% n + 1 100% n  48% 1 10′ final 69% n + 1  95% n 231% ext D1SS13 blue280-294 no final ext 0% n + 1 100% n 101% 0 D16S50 yellow 294-310 nofinal ext 0% n ++01 1 100% n 102% 3 10′ final 73% n + 1 100% n 166% extD15S11 green 316-334 no final ext 17% n + 1 100% n 130% 7 10′ final 77%n + 1 100% n 326% ext D16S51 blue 320-350 no final ext 32% n + 1 100% n486% 5 10′ final 100% 100% n 298% ext n + 1

[0273] The results shown in Table 6 confirm that under conditionsfavoring (“10′ final ext”) or inhibiting (“no final ext”) 3′nontemplated nucleotide addition, Tne DNA polymerase produced PCRproducts that were 95-100% free from non-templated nucleotide addition(“n”) for each locus examined. Taq DNA polymerase, however, demonstratedsignificant addition of nontemplated nucleotides under inhibitingconditions in most loci tested, while under permissive conditions wellover half, and in some cases all, of the PCR product produced by Taq DNApolymerase demonstrated an additional nontemplated 3′ nucleotide.Furthermore, under most conditions the amount of PCR product yielded byTne DNA polymerase was at least as high as that of Taq DNA polymerase,and for some loci was 3- to 4-fold higher.

[0274]FIG. 9 shows two examples of electropherogram gel scans, alignedby PCR product size, comparing the PCR products obtained with Taq andTne polymerases with a 10-minute final extension. For the D15S153 locus,Taq exhibited non-templated nucleotide addition to 40% of the PCRproduct (FIG. 39), while Tne exhibited no such addition of non-templatednucleotides (FIG. 9B). Similar results were obtained with the D15S127locus: 53% of the Taq PCR products demonstrated non-templated nucleotideaddition (FIG. 9C), while none of the Tne PCR products demonstratednon-templated nucleotide addition (FIG. 9D). These results demonstratethe difficulty in identifying alleles in a heterogeneous pattern asgenerated by Taq amplification, compared to the more homogeneous, simplepattern generated by amplification with Tne.

[0275] Together with Examples 24-26, these results indicate that Tne DNApolymerase and the mutants thereof tested in the present studies aresubstantially reduced in the ability to add a nontemplated 3′ terminalnucleotide to DNA templates, particularly DNA templates comprisingmicrosatellite DNA sequences or STRs. Conversely, Taq DNA polymerasedemonstrates significant addition of nontemplated 3′ nucleotides to PCRproducts.

EXAMPLE 28

[0276] Comparison of Taq and Tne

[0277] To examine the ability of a truncated form of Tne DNA polymerase(N′Δ283, 5′exo−, 10% 3′exo activity) to add a nucleotide to the end ofthe PCR product, the enzyme was compared side-by-side with wild type TaqDNA polymerase in amplifications of short tandem repeats at 5 differentmarker loci. A portion of ABI Prism Linkage Mapping Set Panel 21 wasused for the primer sets for the loci. 15 ul reactions (20 mM Tris-HCl,pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 200 uM each dNTP, 333 nM each primer,60 ng human DNA, 0.1% nonionic detergent, 0.6 U DNA polymerase) wereassembled on ice.

[0278] Reactions were loaded into a Perkin Elmer model 9600 thermocyclerpreheated to 95° C. and PCR was done using recommended cyclingconditions (5 min. pre-denaturation at 95° C.; 10 cycles of 15 sec at95° C., 15 sec at 55° C., and 60 sec at 72° C.; 20 cycles of 15 sec at89° C., 15 sec at 55° C., and 60 sec 10min final extension at 72° C.). Aportion of each reaction was diluted, mixed with loading cocktail, heatdenatured and loaded on a 8% sequencing gel. The ABI 373 StretchAutomated Sequencer was run for 5-6hr at 15 W in order to obtain 1 baseresolution. Data was analyzed using GeneScan software. Areas of thepeaks recognized by the software were used to estimate the percent ofextranucleotide addition. Table 7 summarizes the results obtained.Examples of the electropherogram data is shown in FIG. 10. TABLE 7Percent extranucleotide addition exhibited by Taq and Tne DNApolymerases at specific loci. Locus Taq(% n + 1) Tne(% n + 1) D16S405 460 D16S401 100 45 D16S520 63 0 D15S131 51 0 D16S411 53 0

EXAMPLE 29

[0279] Comparison of Tne Mutants

[0280] In order to evaluate the effect of amino acid substitutions inTne DNA polymerase in regard to extra nucleotide addition, differentmutations at position F730 in the untruncated polymerase were comparedin side-by-side amplifications with Taq(wild type) and a truncatedTne(N′Δ219, D323A, F730Y) utilizing a portion of ABI Prism LinkageMapping Set Panel 21. Six loci were examined. 15 ul reactions (20 mMTris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 200 uM each dNTP, 333 nM eachprimer, 50-60 ng human DNA, 0.1% nonionic detergent, 0.15-0.6 U DNApolymerase) were assembled on ice.

[0281] Reactions were loaded into a Perkin Elmer model 9600 thermocyclerpreheated to 95° C. and PCR was done using recommended cyclingconditions (5 min. pre-denaturation at 95° C.; 10 cycles of 15 sec at95° C., 15 sec at 55 ° C., and 60 sec at 72° C.; 20 cycles of 15 sec at89° C., 15 sec at 55° C., and 60 sec at 72° C.; 10 min final extensionat 72° C.). A portion of each reaction was diluted, mixed with loadingcocktail, heat denatured and loaded on a 8% sequencing gel. The ABI 373Stretch Automated Sequencer was run for 5-6 hr at 15 W in order toobtain 1 base resolution. Data was analyzed using GeneScan software.Areas of the peaks recognized by the software were used to estimate thepercent of extranucleotide addition. Table 8 summarizes the resultsobtained. An example of the electropherogram data is shown in FIG. 11.TABLE 8 Percent extranucleotide addition exhibited by mutant Tne DNApolymerases at specific loci. mutant: locus: D16S405 D16S401 D15S131D15S127 D16S511 D15S153 Taq (wild type) 46%  100% 51% 100% 100% 100%Tne-1 (N′Δ219, D323A, F730Y) 0% 0% 0% 0% 0% 0% Tne-35 (D137A, D323A) 0%52% 0% 0% 0% 0% Tne-18 (D137A, D323A,F730Y) 0% 2% 0% 0% 0% 0% Tne-13(D137A, D323A, R722H, 0% 0% 0% 0% 0% 0% F730Y) Tne-14 (D137A, D323A,F730A) nd 0% 0% nd nd nd Tne-47 (D137A, D323A, F730S) 0% 0% 0% 0% 0% 0%Tne-48 (D137A, D323A, F730T) 0% 0% 0% 0% 0% 0%

EXAMPLE 30

[0282] Comparison of Tne and Taq Mutants

[0283] In order to evaluate the effect of amino acid substitution atposition F667 in Taq DNA polymerase(equivalent to F730 in Tne DNApolymerase) in regard to extra nucleotide addition, a commerciallyavailable mutant of Taq DNA polymerase (Taq FS) (N′Δ3, G46D, F667Y) wascompared in side-by-side amplifications with Taq DNA polymerase(wildtype) and Tne-1 DNA polymerase(N′Δ219, D323A, F730Y). Three loci wereexamined (a portion of ABI Prism Linkage Mapping Set Panel 21). 15 ulreactions (20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 200 uM eachdNTP, 333 nM each primer, 60 ng human DNA, 0.1% nonionic detergent, 0.6U DNA polymerase) were assembled on ice.

[0284] Reactions were loaded into a Perkin Elmer model 9600 thermocyclerpreheated to 95° C. and PCR was done using recommended cyclingconditions (5 min. pre-denaturation at 95° C.; 10 cycles of 15 sec at95° C., 15 sec at 55° C., and 60 sec at 72° C.; 20 cycles of 15 sec at89° C., 15 sec at 55° C., and 60 sec at 72° C.; 10 min final extensionat 72° C.). A portion of each reaction was diluted, mixed with loadingcocktail, heat denatured and loaded on a 8% sequencing gel. The ABI 373Stretch Automated Sequencer was run for 5-6 hr at 15 W in order toobtain 1 base resolution. Data was analyzed using GeneScan software.Areas of the peaks recognized by the software were used to estimate thepercent of extranucleotide addition. Table 9 summarizes the resultsobtained. Examples of the electropherogram data are shown in FIG. 12.TABLE 9 Percent extranucleotide addition exhibited by Taq and Tne DNApolymerases at specific loci. locus Taq(% n + 1) TaqFS(% n + 1) Tne-1(%n + 1) D16S411 48 0 0 D15S127 100 31 5 D15S153 100 29 0

EXAMPLE 31

[0285] Comparison of Tne Mutants

[0286] In order to evaluate the effect of amino acid substitutions atposition R722 in Tne DNA polymerase in regard to extranucleotideaddition, different mutations in the polymerase were compared inside-by-side amplifications utilizing a portion of ABI Prism LinkageMapping Set Panel 21. Six loci were examined. 15 ul reactions (20 mMTris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 200 uM each dNTP, 333 nM eachprimer, 50-60 ng human DNA, 0.1% nonionic detergent, 0.2-0.6 U DNApolymerase) were assembled on ice.

[0287] Reactions were loaded into a Perkin Elmer model 9600 thermocyclerpreheated to 95° C. and PCR was done using recommended cyclingconditions (5 min. pre-denaturation at 95° C.; 10 cycles of 15 sec at95° C., 15 sec at 55° C., and 60 sec at 72° C.; 20 cycles of 15 sec at89° C., 15 sec at 55° C., and 60 sec at 72° C.; 10 min final extensionat 72° C.). A portion of each reaction was diluted, mixed with loadingcocktail, heat denatured and loaded on a 8% sequencing gel. The ABI 373Stretch Automated Sequencer was run for 5-6 hr at 15 W in order toobtain lbase resolution. Data was analyzed using GeneScan software.Heights of the n and n+1 peaks recognized by the software were used toestimate the percent of extranucleotide addition. Table 10 summarizesthe results obtained. An example of the electropherogram data is shownin FIG. 13. TABLE 10 Percent extranucleotide addition exhibited bymutant Tne DNA polymerases at specific loci. mutant: locus: D16S405D16S401 DlSSl3l D15S127 D16S511 D15S153 Tne-35 (D137A, D323A) 0% 54%  0%0% 0% 0% Tne-109 (D137A, D323A, R722Y) 0% 0% 0% 0% 0% 0% Tne-110 (D137A,D323A, R722L) 0% 0% 0% 0% 0% 0% Tne-114 (D137A, D323A, R722K) 0% 0% 0%0% 0% 0% Tne-115 (D137A, D323A, R722Q) 0% 0% 0% 0% 0% 0% Tne-116 (D137A,D323A, R722H) 0% 0% 0% 0% 0% 0%

EXAMPLE 32

[0288] Generation of Tne DNA Polymerase Mutant K726R

[0289] The mutation of the Tne polymerase was done by essentially thesame procedure as described above in Example 13. The single-stranded DNAwas isolated from pSport-Tne containing D137A and D323A mutations. Theoligonucleotide used for the mutagenesis was 5′-GAA GTT CAC CAT CCG GCCGAC CCG TCG CAT TTC 3′ (SEQ ID NO:93). An XmaIII site (bold italics inthe above sequence) was introduced into the oligonucleotide for easyscreening of the mutants. The mutation was confirmed by DNA sequencing.The clone was named pTne129 (D137A, D323A, K726R).

EXAMPLE 33

[0290] Determination of the Activity of Non-templated One Base Additionfor Tne DNA Polymerase and its Mutant D137A, D323A, K726R, by PrimerExtension Assay

[0291] The mutant Tne DNA polymerase (Tne D137A, D323A, K726R) preparedin Example 32 was purified as described in Example 20. The assay fornon-templated one base addition was conducted as described in Example23. The results were as follows: Tne DNA Polymerase % of Product WithN + 1 D137A, D323A 78.4 D137A, D323A, R722H 1.7 D137A, D323A, K726R 0.9

[0292] These results demonstrate that mutation of the lysine residue atposition 726 of Tne, particularly to arginine, substantially reduces theactivity of the polymerase in adding non-templated bases.

[0293] Having now fully described the present invention in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious to one of ordinary skill in the artthat the same can be performed by modifying or changing the inventionwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or any specificembodiment thereof, and that such modifications or changes are intendedto be encompassed within the scope of the appended claims.

[0294] All publications, patents and patent applications mentioned inthis specification are indicative of the level of skill of those skilledin the art to which this invention pertains, and are herein incorporatedby reference to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated by reference.

1 93 2682 base pairs nucleic acid both both cDNA 1 ATGGCGAGAC TATTTCTCTTTGATGGCACA GCCCTGGCCT ACAGGGCATA TTACGCCCTC 60 GACAGATCCC TTTCCACATCCACAGGAATT CCAACGAACG CCGTCTATGG CGTTGCCAGG 120 ATGCTCGTTA AATTCATTAAGGAACACATT ATACCCGAAA AGGACTACGC GGCTGTGGCC 180 TTCGACAAGA AGGCAGCGACGTTCAGACAC AAACTGCTCG TAAGCGACAA GGCGCAAAGG 240 CCAAAGACTC CGGCTCTTCTAGTTCAGCAG CTACCTTACA TCAAGCGGCT GATAGAAGCT 300 CTTGGTTTCA AAGTGCTGGAGCTGGAGGGA TACGAAGCAG ACGATATCAT CGCCACGCTT 360 GCAGTCAGGG CTGCACGTTTTTTGATGAGA TTTTCATTAA TAACCGGTGA CAAGGATATG 420 CTTCAACTTG TAAACGAGAAGATAAAGGTC TGGAGAATCG TCAAGGGGAT ATCGGATCTT 480 GAGCTTTACG ATTCGAAAAAGGTGAAAGAA AGATACGGTG TGGAACCACA TCAGATACCG 540 GATCTTCTAG CACTGACGGGAGACGACATA GACAACATTC CCGGTGTAAC GGGAATAGGT 600 GAAAAGACCG CTGTACAGCTTCTCGGCAAG TATAGAAATC TTGAATACAT TCTGGAGCAT 660 GCCCGTGAAC TCCCCCAGAGAGTGAGAAAG GCTCTCTTGA GAGACAGGGA AGTTGCCATC 720 CTCAGTAAAA AACTTGCAACTCTGGTGACG AACGCACCTG TTGAAGTGGA CTGGGAAGAG 780 ATGAAATACA GAGGATACGACAAGAGAAAA CTACTTCCGA TATTGAAAGA ACTGGAGTTT 840 GCTTCCATCA TGAAGGAACTTCAACTGTAC GAAGAAGCAG AACCCACCGG ATACGAAATC 900 GTGAAGGATC ATAAGACCTTCGAAGATCTC ATCGAAAAGC TGAAGGAGGT TCCATCTTTT 960 GCCCTGGACC TTGAAACGTCCTCCCTTGAC CCGTTCAACT GTGAGATAGT CGGCATCTCC 1020 GTGTCGTTCA AACCGAAAACAGCTTATTAC ATTCCACTTC ATCACAGAAA CGCCCAGAAT 1080 CTTGATGAAA CACTGGTGCTGTCGAAGTTG AAAGAGATCC TCGAAGACCC GTCTTCGAAG 1140 ATTGTGGGTC AGAACCTGAAGTACGACTAC AAGGTTCTTA TGGTAAAGGG TATATCGCCA 1200 GTTTATCCGC ATTTTGACACGATGATAGCT GCATATTTGC TGGAGCCAAA CGAGAAAAAA 1260 TTCAATCTCG AAGATCTGTCTTTGAAATTT CTCGGATACA AAATGACGTC TTATCAGGAA 1320 CTGATGTCGT TTTCCTCACCACTTTTTGGT TTCAGCTTTG CGGATGTTCC GGTAGACAAG 1380 GCTGCGAACT ACTCCTGCGAGGATGCAGAC ATCACTTATA GGCTCTACAA GATACTCAGC 1440 ATGAAGCTCC ATGAAGCGGAACTTGAGAAC GTCTTCTACA GGATAGAGAT GCCGTTGGTG 1500 AACGTTCTTG CACGCATGGAATTGAACGGG GTGTATGTGG ACACAGAATT CCTGAAAAAG 1560 CTCTCGGAGG AGTACGGCAAAAAGCTCGAG GAACTGGCCG AAAAAATCTA CCAGATAGCA 1620 GGTGAGCCCT TCAACATCAATTCTCCAAAA CAGGTTTCAA AGATCCTTTT TGAGAAGCTG 1680 GGAATAAAAC CCCGTGGAAAAACGACAAAA ACAGGAGAGT ACTCTACCAG GATAGAGGTG 1740 TTGGAAGAGA TAGCGAATGAGCACGAGATA GTACCCCTCA TTCTCGAGTA CAGAAAGATC 1800 CAGAAACTGA AATCGACCTACATAGACACC CTTCCGAAAC TTGTGAACCC GAAAACCGGA 1860 AGAATTCATG CATCTTTCCACCAGACGGGT ACCGCCACTG GCAGGTTGAG TAGCAGTGAT 1920 CCAAATCTTC AGAATCTTCCGACAAAGAGC GAAGAGGGAA AAGAAATTAG AAAAGCGATT 1980 GTGCCCCAGG ATCCAGACTGGTGGATCGTC AGTGCGGATT ATTCCCAAAT AGAACTCAGA 2040 ATCCTCGCTC ATCTCAGTGGTGATGAGAAC CTTGTGAAGG CCTTCGAGGA GGGCATCGAT 2100 GTGCACACCT TGACTGCCTCCAGGATCTAC AACGTAAAGC CAGAAGAAGT GAACGAAGAA 2160 ATGCGACGGG TTGGAAAGATGGTGAACTTC TCTATAATAT ACGGTGTCAC ACCGTACGGT 2220 CTTTCTGTGA GACTTGGAATACCGGTTAAA GAAGCAGAAA AGATGATTAT CAGCTATTTC 2280 ACACTGTATC CAAAGGTGCGAAGCTACATC CAGCAGGTTG TTGCAGAGGC AAAAGAGAAG 2340 GGCTACGTCA GGACTCTCTTTGGAAGAAAA AGAGATATTC CCCAGCTCAT GGCAAGGGAC 2400 AAGAACACCC AGTCCGAAGGCGAAAGAATC GCAATAAACA CCCCCATTCA GGGAACGGCG 2460 GCAGATATAA TAAAATTGGCTATGATAGAT ATAGACGAGG AGCTGAGAAA AAGAAACATG 2520 AAATCCAGAA TGATCATTCAGGTTCATGAC GAACTGGTCT TCGAGGTTCC CGATGAGGAA 2580 AAAGAAGAAC TAGTTGATCTGGTGAAGAAC AAAATGACAA ATGTGGTGAA ACTCTCTGTG 2640 CCTCTTGAGG TTGACATAAGCATCGGAAAA AGCTGGTCTT GA 2682 893 amino acids amino acid not relevantnot relevant protein 2 Met Ala Arg Leu Phe Leu Phe Asp Gly Thr Ala LeuAla Tyr Arg Ala 1 5 10 15 Tyr Tyr Ala Leu Asp Arg Ser Leu Ser Thr SerThr Gly Ile Pro Thr 20 25 30 Asn Ala Val Tyr Gly Val Ala Arg Met Leu ValLys Phe Ile Lys Glu 35 40 45 His Ile Ile Pro Glu Lys Asp Tyr Ala Ala ValAla Phe Asp Lys Lys 50 55 60 Ala Ala Thr Phe Arg His Lys Leu Leu Val SerAsp Lys Ala Gln Arg 65 70 75 80 Pro Lys Thr Pro Ala Leu Leu Val Gln GlnLeu Pro Tyr Ile Lys Arg 85 90 95 Leu Ile Glu Ala Leu Gly Phe Lys Val LeuGlu Leu Glu Gly Tyr Glu 100 105 110 Ala Asp Asp Ile Ile Ala Thr Leu AlaVal Arg Ala Ala Arg Phe Leu 115 120 125 Met Arg Phe Ser Leu Ile Thr GlyAsp Lys Asp Met Leu Gln Leu Val 130 135 140 Asn Glu Lys Ile Lys Val TrpArg Ile Val Lys Gly Ile Ser Asp Leu 145 150 155 160 Glu Leu Tyr Asp SerLys Lys Val Lys Glu Arg Tyr Gly Val Glu Pro 165 170 175 His Gln Ile ProAsp Leu Leu Ala Leu Thr Gly Asp Asp Ile Asp Asn 180 185 190 Ile Pro GlyVal Thr Gly Ile Gly Glu Lys Thr Ala Val Gln Leu Leu 195 200 205 Gly LysTyr Arg Asn Leu Glu Tyr Ile Leu Glu His Ala Arg Glu Leu 210 215 220 ProGln Arg Val Arg Lys Ala Leu Leu Arg Asp Arg Glu Val Ala Ile 225 230 235240 Leu Ser Lys Lys Leu Ala Thr Leu Val Thr Asn Ala Pro Val Glu Val 245250 255 Asp Trp Glu Glu Met Lys Tyr Arg Gly Tyr Asp Lys Arg Lys Leu Leu260 265 270 Pro Ile Leu Lys Glu Leu Glu Phe Ala Ser Ile Met Lys Glu LeuGln 275 280 285 Leu Tyr Glu Glu Ala Glu Pro Thr Gly Tyr Glu Ile Val LysAsp His 290 295 300 Lys Thr Phe Glu Asp Leu Ile Glu Lys Leu Lys Glu ValPro Ser Phe 305 310 315 320 Ala Leu Asp Leu Glu Thr Ser Ser Leu Asp ProPhe Asn Cys Glu Ile 325 330 335 Val Gly Ile Ser Val Ser Phe Lys Pro LysThr Ala Tyr Tyr Ile Pro 340 345 350 Leu His His Arg Asn Ala Gln Asn LeuAsp Glu Thr Leu Val Leu Ser 355 360 365 Lys Leu Lys Glu Ile Leu Glu AspPro Ser Ser Lys Ile Val Gly Gln 370 375 380 Asn Leu Lys Tyr Asp Tyr LysVal Leu Met Val Lys Gly Ile Ser Pro 385 390 395 400 Val Tyr Pro His PheAsp Thr Met Ile Ala Ala Tyr Leu Leu Glu Pro 405 410 415 Asn Glu Lys LysPhe Asn Leu Glu Asp Leu Ser Leu Lys Phe Leu Gly 420 425 430 Tyr Lys MetThr Ser Tyr Gln Glu Leu Met Ser Phe Ser Ser Pro Leu 435 440 445 Phe GlyPhe Ser Phe Ala Asp Val Pro Val Asp Lys Ala Ala Asn Tyr 450 455 460 SerCys Glu Asp Ala Asp Ile Thr Tyr Arg Leu Tyr Lys Ile Leu Ser 465 470 475480 Met Lys Leu His Glu Ala Glu Leu Glu Asn Val Phe Tyr Arg Ile Glu 485490 495 Met Pro Leu Val Asn Val Leu Ala Arg Met Glu Leu Asn Gly Val Tyr500 505 510 Val Asp Thr Glu Phe Leu Lys Lys Leu Ser Glu Glu Tyr Gly LysLys 515 520 525 Leu Glu Glu Leu Ala Glu Lys Ile Tyr Gln Ile Ala Gly GluPro Phe 530 535 540 Asn Ile Asn Ser Pro Lys Gln Val Ser Lys Ile Leu PheGlu Lys Leu 545 550 555 560 Gly Ile Lys Pro Arg Gly Lys Thr Thr Lys ThrGly Glu Tyr Ser Thr 565 570 575 Arg Ile Glu Val Leu Glu Glu Ile Ala AsnGlu His Glu Ile Val Pro 580 585 590 Leu Ile Leu Glu Tyr Arg Lys Ile GlnLys Leu Lys Ser Thr Tyr Ile 595 600 605 Asp Thr Leu Pro Lys Leu Val AsnPro Lys Thr Gly Arg Ile His Ala 610 615 620 Ser Phe His Gln Thr Gly ThrAla Thr Gly Arg Leu Ser Ser Ser Asp 625 630 635 640 Pro Asn Leu Gln AsnLeu Pro Thr Lys Ser Glu Glu Gly Lys Glu Ile 645 650 655 Arg Lys Ala IleVal Pro Gln Asp Pro Asp Trp Trp Ile Val Ser Ala 660 665 670 Asp Tyr SerGln Ile Glu Leu Arg Ile Leu Ala His Leu Ser Gly Asp 675 680 685 Glu AsnLeu Val Lys Ala Phe Glu Glu Gly Ile Asp Val His Thr Leu 690 695 700 ThrAla Ser Arg Ile Tyr Asn Val Lys Pro Glu Glu Val Asn Glu Glu 705 710 715720 Met Arg Arg Val Gly Lys Met Val Asn Phe Ser Ile Ile Tyr Gly Val 725730 735 Thr Pro Tyr Gly Leu Ser Val Arg Leu Gly Ile Pro Val Lys Glu Ala740 745 750 Glu Lys Met Ile Ile Ser Tyr Phe Thr Leu Tyr Pro Lys Val ArgSer 755 760 765 Tyr Ile Gln Gln Val Val Ala Glu Ala Lys Glu Lys Gly TyrVal Arg 770 775 780 Thr Leu Phe Gly Arg Lys Arg Asp Ile Pro Gln Leu MetAla Arg Asp 785 790 795 800 Lys Asn Thr Gln Ser Glu Gly Glu Arg Ile AlaIle Asn Thr Pro Ile 805 810 815 Gln Gly Thr Ala Ala Asp Ile Ile Lys LeuAla Met Ile Asp Ile Asp 820 825 830 Glu Glu Leu Arg Lys Arg Asn Met LysSer Arg Met Ile Ile Gln Val 835 840 845 His Asp Glu Leu Val Phe Glu ValPro Asp Glu Glu Lys Glu Glu Leu 850 855 860 Val Asp Leu Val Lys Asn LysMet Thr Asn Val Val Lys Leu Ser Val 865 870 875 880 Pro Leu Glu Val AspIle Ser Ile Gly Lys Ser Trp Ser 885 890 677 amino acids amino acid notrelevant not relevant protein 3 Met Ser Leu His Ala Arg Glu Leu Pro GlnArg Val Arg Lys Ala Leu 1 5 10 15 Leu Arg Asp Arg Glu Val Ala Ile LeuSer Lys Lys Leu Ala Thr Leu 20 25 30 Val Thr Asn Ala Pro Val Glu Val AspTrp Glu Glu Met Lys Tyr Arg 35 40 45 Gly Tyr Asp Lys Arg Lys Leu Leu ProIle Leu Lys Glu Leu Glu Phe 50 55 60 Ala Ser Ile Met Lys Glu Leu Gln LeuTyr Glu Glu Ala Glu Pro Thr 65 70 75 80 Gly Tyr Glu Ile Val Lys Asp HisLys Thr Phe Glu Asp Leu Ile Glu 85 90 95 Lys Leu Lys Glu Val Pro Ser PheAla Leu Ala Leu Glu Thr Ser Ser 100 105 110 Leu Asp Pro Phe Asn Cys GluIle Val Gly Ile Ser Val Ser Phe Lys 115 120 125 Pro Lys Thr Ala Tyr TyrIle Pro Leu His His Arg Asn Ala Gln Asn 130 135 140 Leu Asp Glu Thr LeuVal Leu Ser Lys Leu Lys Glu Ile Leu Glu Asp 145 150 155 160 Pro Ser SerLys Ile Val Gly Gln Asn Leu Lys Tyr Asp Tyr Lys Val 165 170 175 Leu MetVal Lys Gly Ile Ser Pro Val Tyr Pro His Phe Asp Thr Met 180 185 190 IleAla Ala Tyr Leu Leu Glu Pro Asn Glu Lys Lys Phe Asn Leu Glu 195 200 205Asp Leu Ser Leu Lys Phe Leu Gly Tyr Lys Met Thr Ser Tyr Gln Glu 210 215220 Leu Met Ser Phe Ser Ser Pro Leu Phe Gly Phe Ser Phe Ala Asp Val 225230 235 240 Pro Val Asp Lys Ala Ala Asn Tyr Ser Cys Glu Asp Ala Asp IleThr 245 250 255 Tyr Arg Leu Tyr Lys Ile Leu Ser Met Lys Leu His Glu AlaGlu Leu 260 265 270 Glu Asn Val Phe Tyr Arg Ile Glu Met Pro Leu Val AsnVal Leu Ala 275 280 285 Arg Met Glu Leu Asn Gly Val Tyr Val Asp Thr GluPhe Leu Lys Lys 290 295 300 Leu Ser Glu Glu Tyr Gly Lys Lys Leu Glu GluLeu Ala Glu Lys Ile 305 310 315 320 Tyr Gln Ile Ala Gly Glu Pro Phe AsnIle Asn Ser Pro Lys Gln Val 325 330 335 Ser Lys Ile Leu Phe Glu Lys LeuGly Ile Lys Pro Arg Gly Lys Thr 340 345 350 Thr Lys Thr Gly Glu Tyr SerThr Arg Ile Glu Val Leu Glu Glu Ile 355 360 365 Ala Asn Glu His Glu IleVal Pro Leu Ile Leu Glu Tyr Arg Lys Ile 370 375 380 Gln Lys Leu Lys SerThr Tyr Ile Asp Thr Leu Pro Lys Leu Val Asn 385 390 395 400 Pro Lys ThrGly Arg Ile His Ala Ser Phe His Gln Thr Gly Thr Ala 405 410 415 Thr GlyArg Leu Ser Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr 420 425 430 LysSer Glu Glu Gly Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp 435 440 445Pro Asp Trp Trp Ile Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg 450 455460 Ile Leu Ala His Leu Ser Gly Asp Glu Asn Leu Val Lys Ala Phe Glu 465470 475 480 Glu Gly Ile Asp Val His Thr Leu Thr Ala Ser Arg Ile Tyr AsnVal 485 490 495 Lys Pro Glu Glu Val Asn Glu Glu Met Arg Arg Val Gly LysMet Val 500 505 510 Asn Phe Ser Ile Ile Tyr Gly Val Thr Pro Tyr Gly LeuSer Val Arg 515 520 525 Leu Gly Ile Pro Val Lys Glu Ala Glu Lys Met IleIle Ser Tyr Phe 530 535 540 Thr Leu Tyr Pro Lys Val Arg Ser Tyr Ile GlnGln Val Val Ala Glu 545 550 555 560 Ala Lys Glu Lys Gly Tyr Val Arg ThrLeu Phe Gly Arg Lys Arg Asp 565 570 575 Ile Pro Gln Leu Met Ala Arg AspLys Asn Thr Gln Ser Glu Gly Glu 580 585 590 Arg Ile Ala Ile Asn Thr ProIle Gln Gly Thr Ala Ala Asp Ile Ile 595 600 605 Lys Leu Ala Met Ile AspIle Asp Glu Glu Leu Arg Lys Arg Asn Met 610 615 620 Lys Ser Arg Met IleIle Gln Val His Asp Glu Leu Val Phe Glu Val 625 630 635 640 Pro Asp GluGlu Lys Glu Glu Leu Val Asp Leu Val Lys Asn Lys Met 645 650 655 Thr AsnVal Val Lys Leu Ser Val Pro Leu Glu Val Asp Ile Ser Ile 660 665 670 GlyLys Ser Trp Ser 675 610 amino acids amino acid not relevant not relevantprotein 4 Met Lys Glu Leu Gln Leu Tyr Glu Glu Ala Glu Pro Thr Gly TyrGlu 1 5 10 15 Ile Val Lys Asp His Lys Thr Phe Glu Asp Leu Ile Glu LysLeu Lys 20 25 30 Glu Val Pro Ser Phe Ala Leu Ala Leu Glu Thr Ser Ser LeuAsp Pro 35 40 45 Phe Asn Cys Glu Ile Val Gly Ile Ser Val Ser Phe Lys ProLys Thr 50 55 60 Ala Tyr Tyr Ile Pro Leu His His Arg Asn Ala Gln Asn LeuAsp Glu 65 70 75 80 Thr Leu Val Leu Ser Lys Leu Lys Glu Ile Leu Glu AspPro Ser Ser 85 90 95 Lys Ile Val Gly Gln Asn Leu Lys Tyr Asp Tyr Lys ValLeu Met Val 100 105 110 Lys Gly Ile Ser Pro Val Tyr Pro His Phe Asp ThrMet Ile Ala Ala 115 120 125 Tyr Leu Leu Glu Pro Asn Glu Lys Lys Phe AsnLeu Glu Asp Leu Ser 130 135 140 Leu Lys Phe Leu Gly Tyr Lys Met Thr SerTyr Gln Glu Leu Met Ser 145 150 155 160 Phe Ser Ser Pro Leu Phe Gly PheSer Phe Ala Asp Val Pro Val Asp 165 170 175 Lys Ala Ala Asn Tyr Ser CysGlu Asp Ala Asp Ile Thr Tyr Arg Leu 180 185 190 Tyr Lys Ile Leu Ser MetLys Leu His Glu Ala Glu Leu Glu Asn Val 195 200 205 Phe Tyr Arg Ile GluMet Pro Leu Val Asn Val Leu Ala Arg Met Glu 210 215 220 Leu Asn Gly ValTyr Val Asp Thr Glu Phe Leu Lys Lys Leu Ser Glu 225 230 235 240 Glu TyrGly Lys Lys Leu Glu Glu Leu Ala Glu Lys Ile Tyr Gln Ile 245 250 255 AlaGly Glu Pro Phe Asn Ile Asn Ser Pro Lys Gln Val Ser Lys Ile 260 265 270Leu Phe Glu Lys Leu Gly Ile Lys Pro Arg Gly Lys Thr Thr Lys Thr 275 280285 Gly Glu Tyr Ser Thr Arg Ile Glu Val Leu Glu Glu Ile Ala Asn Glu 290295 300 His Glu Ile Val Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu305 310 315 320 Lys Ser Thr Tyr Ile Asp Thr Leu Pro Lys Leu Val Asn ProLys Thr 325 330 335 Gly Arg Ile His Ala Ser Phe His Gln Thr Gly Thr AlaThr Gly Arg 340 345 350 Leu Ser Ser Ser Asp Pro Asn Leu Gln Asn Leu ProThr Lys Ser Glu 355 360 365 Glu Gly Lys Glu Ile Arg Lys Ala Ile Val ProGln Asp Pro Asp Trp 370 375 380 Trp Ile Val Ser Ala Asp Tyr Ser Gln IleGlu Leu Arg Ile Leu Ala 385 390 395 400 His Leu Ser Gly Asp Glu Asn LeuVal Lys Ala Phe Glu Glu Gly Ile 405 410 415 Asp Val His Thr Leu Thr AlaSer Arg Ile Tyr Asn Val Lys Pro Glu 420 425 430 Glu Val Asn Glu Glu MetArg Arg Val Gly Lys Met Val Asn Phe Ser 435 440 445 Ile Ile Tyr Gly ValThr Pro Tyr Gly Leu Ser Val Arg Leu Gly Ile 450 455 460 Pro Val Lys GluAla Glu Lys Met Ile Ile Ser Tyr Phe Thr Leu Tyr 465 470 475 480 Pro LysVal Arg Ser Tyr Ile Gln Gln Val Val Ala Glu Ala Lys Glu 485 490 495 LysGly Tyr Val Arg Thr Leu Phe Gly Arg Lys Arg Asp Ile Pro Gln 500 505 510Leu Met Ala Arg Asp Lys Asn Thr Gln Ser Glu Gly Glu Arg Ile Ala 515 520525 Ile Asn Thr Pro Ile Gln Gly Thr Ala Ala Asp Ile Ile Lys Leu Ala 530535 540 Met Ile Asp Ile Asp Glu Glu Leu Arg Lys Arg Asn Met Lys Ser Arg545 550 555 560 Met Ile Ile Gln Val His Asp Glu Leu Val Phe Glu Val ProAsp Glu 565 570 575 Glu Lys Glu Glu Leu Val Asp Leu Val Lys Asn Lys MetThr Asn Val 580 585 590 Val Lys Leu Ser Val Pro Leu Glu Val Asp Ile SerIle Gly Lys Ser 595 600 605 Trp Ser 610 708 amino acids amino acid notrelevant not relevant protein 5 Met Asn Ser Ser Ser Val Pro Ile Pro GlyVal Thr Gly Ile Gly Glu 1 5 10 15 Lys Thr Ala Val Gln Leu Leu Gly LysTyr Arg Asn Leu Glu Tyr Ile 20 25 30 Leu Glu His Ala Arg Glu Leu Pro GlnArg Val Arg Lys Ala Leu Leu 35 40 45 Arg Asp Arg Glu Val Ala Ile Leu SerLys Lys Leu Ala Thr Leu Val 50 55 60 Thr Asn Ala Pro Val Glu Val Asp TrpGlu Glu Met Lys Tyr Arg Gly 65 70 75 80 Tyr Asp Lys Arg Lys Leu Leu ProIle Leu Lys Glu Leu Glu Phe Ala 85 90 95 Ser Ile Met Lys Glu Leu Gln LeuTyr Glu Glu Ala Glu Pro Thr Gly 100 105 110 Tyr Glu Ile Val Lys Asp HisLys Thr Phe Glu Asp Leu Ile Glu Lys 115 120 125 Leu Lys Glu Val Pro SerPhe Ala Leu Ala Leu Glu Thr Ser Ser Leu 130 135 140 Asp Pro Phe Asn CysGlu Ile Val Gly Ile Ser Val Ser Phe Lys Pro 145 150 155 160 Lys Thr AlaTyr Tyr Ile Pro Leu His His Arg Asn Ala Gln Asn Leu 165 170 175 Asp GluThr Leu Val Leu Ser Lys Leu Lys Glu Ile Leu Glu Asp Pro 180 185 190 SerSer Lys Ile Val Gly Gln Asn Leu Lys Tyr Asp Tyr Lys Val Leu 195 200 205Met Val Lys Gly Ile Ser Pro Val Tyr Pro His Phe Asp Thr Met Ile 210 215220 Ala Ala Tyr Leu Leu Glu Pro Asn Glu Lys Lys Phe Asn Leu Glu Asp 225230 235 240 Leu Ser Leu Lys Phe Leu Gly Tyr Lys Met Thr Ser Tyr Gln GluLeu 245 250 255 Met Ser Phe Ser Ser Pro Leu Phe Gly Phe Ser Phe Ala AspVal Pro 260 265 270 Val Asp Lys Ala Ala Asn Tyr Ser Cys Glu Asp Ala AspIle Thr Tyr 275 280 285 Arg Leu Tyr Lys Ile Leu Ser Met Lys Leu His GluAla Glu Leu Glu 290 295 300 Asn Val Phe Tyr Arg Ile Glu Met Pro Leu ValAsn Val Leu Ala Arg 305 310 315 320 Met Glu Leu Asn Gly Val Tyr Val AspThr Glu Phe Leu Lys Lys Leu 325 330 335 Ser Glu Glu Tyr Gly Lys Lys LeuGlu Glu Leu Ala Glu Lys Ile Tyr 340 345 350 Gln Ile Ala Gly Glu Pro PheAsn Ile Asn Ser Pro Lys Gln Val Ser 355 360 365 Lys Ile Leu Phe Glu LysLeu Gly Ile Lys Pro Arg Gly Lys Thr Thr 370 375 380 Lys Thr Gly Glu TyrSer Thr Arg Ile Glu Val Leu Glu Glu Ile Ala 385 390 395 400 Asn Glu HisGlu Ile Val Pro Leu Ile Leu Glu Tyr Arg Lys Ile Gln 405 410 415 Lys LeuLys Ser Thr Tyr Ile Asp Thr Leu Pro Lys Leu Val Asn Pro 420 425 430 LysThr Gly Arg Ile His Ala Ser Phe His Gln Thr Gly Thr Ala Thr 435 440 445Gly Arg Leu Ser Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys 450 455460 Ser Glu Glu Gly Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro 465470 475 480 Asp Trp Trp Ile Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu ArgIle 485 490 495 Leu Ala His Leu Ser Gly Asp Glu Asn Leu Val Lys Ala PheGlu Glu 500 505 510 Gly Ile Asp Val His Thr Leu Thr Ala Ser Arg Ile TyrAsn Val Lys 515 520 525 Pro Glu Glu Val Asn Glu Glu Met Arg Arg Val GlyLys Met Val Asn 530 535 540 Phe Ser Ile Ile Tyr Gly Val Thr Pro Tyr GlyLeu Ser Val Arg Leu 545 550 555 560 Gly Ile Pro Val Lys Glu Ala Glu LysMet Ile Ile Ser Tyr Phe Thr 565 570 575 Leu Tyr Pro Lys Val Arg Ser TyrIle Gln Gln Val Val Ala Glu Ala 580 585 590 Lys Glu Lys Gly Tyr Val ArgThr Leu Phe Gly Arg Lys Arg Asp Ile 595 600 605 Pro Gln Leu Met Ala ArgAsp Lys Asn Thr Gln Ser Glu Gly Glu Arg 610 615 620 Ile Ala Ile Asn ThrPro Ile Gln Gly Thr Ala Ala Asp Ile Ile Lys 625 630 635 640 Leu Ala MetIle Asp Ile Asp Glu Glu Leu Arg Lys Arg Asn Met Lys 645 650 655 Ser ArgMet Ile Ile Gln Val His Asp Glu Leu Val Phe Glu Val Pro 660 665 670 AspGlu Glu Lys Glu Glu Leu Val Asp Leu Val Lys Asn Lys Met Thr 675 680 685Asn Val Val Lys Leu Ser Val Pro Leu Glu Val Asp Ile Ser Ile Gly 690 695700 Lys Ser Trp Ser 705 893 amino acids amino acid not relevant notrelevant peptide 6 Met Ala Arg Leu Phe Leu Phe Asp Gly Thr Ala Leu AlaTyr Arg Ala 1 5 10 15 Tyr Tyr Ala Leu Asp Arg Ser Leu Ser Thr Ser ThrGly Ile Pro Thr 20 25 30 Asn Ala Val Tyr Gly Val Ala Arg Met Leu Val LysPhe Ile Lys Glu 35 40 45 His Ile Ile Pro Glu Lys Asp Tyr Ala Ala Val AlaPhe Asp Lys Lys 50 55 60 Ala Ala Thr Phe Arg His Lys Leu Leu Val Ser AspLys Ala Gln Arg 65 70 75 80 Pro Lys Thr Pro Ala Leu Leu Val Gln Gln LeuPro Tyr Ile Lys Arg 85 90 95 Leu Ile Glu Ala Leu Gly Phe Lys Val Leu GluLeu Glu Gly Tyr Glu 100 105 110 Ala Asp Asp Ile Ile Ala Thr Leu Ala ValArg Ala Ala Arg Phe Leu 115 120 125 Met Arg Phe Ser Leu Ile Thr Gly AlaLys Asp Met Leu Gln Leu Val 130 135 140 Asn Glu Lys Ile Lys Val Trp ArgIle Val Lys Gly Ile Ser Asp Leu 145 150 155 160 Glu Leu Tyr Asp Ser LysLys Val Lys Glu Arg Tyr Gly Val Glu Pro 165 170 175 His Gln Ile Pro AspLeu Leu Ala Leu Thr Gly Asp Asp Ile Asp Asn 180 185 190 Ile Pro Gly ValThr Gly Ile Gly Glu Lys Thr Ala Val Gln Leu Leu 195 200 205 Gly Lys TyrArg Asn Leu Glu Tyr Ile Leu Glu His Ala Arg Glu Leu 210 215 220 Pro GlnArg Val Arg Lys Ala Leu Leu Arg Asp Arg Glu Val Ala Ile 225 230 235 240Leu Ser Lys Lys Leu Ala Thr Leu Val Thr Asn Ala Pro Val Glu Val 245 250255 Asp Trp Glu Glu Met Lys Tyr Arg Gly Tyr Asp Lys Arg Lys Leu Leu 260265 270 Pro Ile Leu Lys Glu Leu Glu Phe Ala Ser Ile Met Lys Glu Leu Gln275 280 285 Leu Tyr Glu Glu Ala Glu Pro Thr Gly Tyr Glu Ile Val Lys AspHis 290 295 300 Lys Thr Phe Glu Asp Leu Ile Glu Lys Leu Lys Glu Val ProSer Phe 305 310 315 320 Ala Leu Ala Leu Glu Thr Ser Ser Leu Asp Pro PheAsn Cys Glu Ile 325 330 335 Val Gly Ile Ser Val Ser Phe Lys Pro Lys ThrAla Tyr Tyr Ile Pro 340 345 350 Leu His His Arg Asn Ala Gln Asn Leu AspGlu Thr Leu Val Leu Ser 355 360 365 Lys Leu Lys Glu Ile Leu Glu Asp ProSer Ser Lys Ile Val Gly Gln 370 375 380 Asn Leu Lys Tyr Asp Tyr Lys ValLeu Met Val Lys Gly Ile Ser Pro 385 390 395 400 Val Tyr Pro His Phe AspThr Met Ile Ala Ala Tyr Leu Leu Glu Pro 405 410 415 Asn Glu Lys Lys PheAsn Leu Glu Asp Leu Ser Leu Lys Phe Leu Gly 420 425 430 Tyr Lys Met ThrSer Tyr Gln Glu Leu Met Ser Phe Ser Ser Pro Leu 435 440 445 Phe Gly PheSer Phe Ala Asp Val Pro Val Asp Lys Ala Ala Asn Tyr 450 455 460 Ser CysGlu Asp Ala Asp Ile Thr Tyr Arg Leu Tyr Lys Ile Leu Ser 465 470 475 480Met Lys Leu His Glu Ala Glu Leu Glu Asn Val Phe Tyr Arg Ile Glu 485 490495 Met Pro Leu Val Asn Val Leu Ala Arg Met Glu Leu Asn Gly Val Tyr 500505 510 Val Asp Thr Glu Phe Leu Lys Lys Leu Ser Glu Glu Tyr Gly Lys Lys515 520 525 Leu Glu Glu Leu Ala Glu Lys Ile Tyr Gln Ile Ala Gly Glu ProPhe 530 535 540 Asn Ile Asn Ser Pro Lys Gln Val Ser Lys Ile Leu Phe GluLys Leu 545 550 555 560 Gly Ile Lys Pro Arg Gly Lys Thr Thr Lys Thr GlyGlu Tyr Ser Thr 565 570 575 Arg Ile Glu Val Leu Glu Glu Ile Ala Asn GluHis Glu Ile Val Pro 580 585 590 Leu Ile Leu Glu Tyr Arg Lys Ile Gln LysLeu Lys Ser Thr Tyr Ile 595 600 605 Asp Thr Leu Pro Lys Leu Val Asn ProLys Thr Gly Arg Ile His Ala 610 615 620 Ser Phe His Gln Thr Gly Thr AlaThr Gly Arg Leu Ser Ser Ser Asp 625 630 635 640 Pro Asn Leu Gln Asn LeuPro Thr Lys Ser Glu Glu Gly Lys Glu Ile 645 650 655 Arg Lys Ala Ile ValPro Gln Asp Pro Asp Trp Trp Ile Val Ser Ala 660 665 670 Asp Tyr Ser GlnIle Glu Leu Arg Ile Leu Ala His Leu Ser Gly Asp 675 680 685 Glu Asn LeuVal Lys Ala Phe Glu Glu Gly Ile Asp Val His Thr Leu 690 695 700 Thr AlaSer Arg Ile Tyr Asn Val Lys Pro Glu Glu Val Asn Glu Glu 705 710 715 720Met Arg Arg Val Gly Lys Met Val Asn Phe Ser Ile Ile Tyr Gly Val 725 730735 Thr Pro Tyr Gly Leu Ser Val Arg Leu Gly Ile Pro Val Lys Glu Ala 740745 750 Glu Lys Met Ile Ile Ser Tyr Phe Thr Leu Tyr Pro Lys Val Arg Ser755 760 765 Tyr Ile Gln Gln Val Val Ala Glu Ala Lys Glu Lys Gly Tyr ValArg 770 775 780 Thr Leu Phe Gly Arg Lys Arg Asp Ile Pro Gln Leu Met AlaArg Asp 785 790 795 800 Lys Asn Thr Gln Ser Glu Gly Glu Arg Ile Ala IleAsn Thr Pro Ile 805 810 815 Gln Gly Thr Ala Ala Asp Ile Ile Lys Leu AlaMet Ile Asp Ile Asp 820 825 830 Glu Glu Leu Arg Lys Arg Asn Met Lys SerArg Met Ile Ile Gln Val 835 840 845 His Asp Glu Leu Val Phe Glu Val ProAsp Glu Glu Lys Glu Glu Leu 850 855 860 Val Asp Leu Val Lys Asn Lys MetThr Asn Val Val Lys Leu Ser Val 865 870 875 880 Pro Leu Glu Val Asp IleSer Ile Gly Lys Ser Trp Ser 885 890 893 amino acids amino acid notrelevant not relevant protein 7 Met Ala Arg Leu Phe Leu Phe Ala Gly ThrAla Leu Ala Tyr Arg Ala 1 5 10 15 Tyr Tyr Ala Leu Asp Arg Ser Leu SerThr Ser Thr Gly Ile Pro Thr 20 25 30 Asn Ala Val Tyr Gly Val Ala Arg MetLeu Val Lys Phe Ile Lys Glu 35 40 45 His Ile Ile Pro Glu Lys Asp Tyr AlaAla Val Ala Phe Asp Lys Lys 50 55 60 Ala Ala Thr Phe Arg His Lys Leu LeuVal Ser Asp Lys Ala Gln Arg 65 70 75 80 Pro Lys Thr Pro Ala Leu Leu ValGln Gln Leu Pro Tyr Ile Lys Arg 85 90 95 Leu Ile Glu Ala Leu Gly Phe LysVal Leu Glu Leu Glu Gly Tyr Glu 100 105 110 Ala Asp Asp Ile Ile Ala ThrLeu Ala Val Arg Ala Ala Arg Phe Leu 115 120 125 Met Arg Phe Ser Leu IleThr Gly Asp Lys Asp Met Leu Gln Leu Val 130 135 140 Asn Glu Lys Ile LysVal Trp Arg Ile Val Lys Gly Ile Ser Asp Leu 145 150 155 160 Glu Leu TyrAsp Ser Lys Lys Val Lys Glu Arg Tyr Gly Val Glu Pro 165 170 175 His GlnIle Pro Asp Leu Leu Ala Leu Thr Gly Asp Asp Ile Asp Asn 180 185 190 IlePro Gly Val Thr Gly Ile Gly Glu Lys Thr Ala Val Gln Leu Leu 195 200 205Gly Lys Tyr Arg Asn Leu Glu Tyr Ile Leu Glu His Ala Arg Glu Leu 210 215220 Pro Gln Arg Val Arg Lys Ala Leu Leu Arg Asp Arg Glu Val Ala Ile 225230 235 240 Leu Ser Lys Lys Leu Ala Thr Leu Val Thr Asn Ala Pro Val GluVal 245 250 255 Asp Trp Glu Glu Met Lys Tyr Arg Gly Tyr Asp Lys Arg LysLeu Leu 260 265 270 Pro Ile Leu Lys Glu Leu Glu Phe Ala Ser Ile Met LysGlu Leu Gln 275 280 285 Leu Tyr Glu Glu Ala Glu Pro Thr Gly Tyr Glu IleVal Lys Asp His 290 295 300 Lys Thr Phe Glu Asp Leu Ile Glu Lys Leu LysGlu Val Pro Ser Phe 305 310 315 320 Ala Leu Ala Leu Glu Thr Ser Ser LeuAsp Pro Phe Asn Cys Glu Ile 325 330 335 Val Gly Ile Ser Val Ser Phe LysPro Lys Thr Ala Tyr Tyr Ile Pro 340 345 350 Leu His His Arg Asn Ala GlnAsn Leu Asp Glu Thr Leu Val Leu Ser 355 360 365 Lys Leu Lys Glu Ile LeuGlu Asp Pro Ser Ser Lys Ile Val Gly Gln 370 375 380 Asn Leu Lys Tyr AspTyr Lys Val Leu Met Val Lys Gly Ile Ser Pro 385 390 395 400 Val Tyr ProHis Phe Asp Thr Met Ile Ala Ala Tyr Leu Leu Glu Pro 405 410 415 Asn GluLys Lys Phe Asn Leu Glu Asp Leu Ser Leu Lys Phe Leu Gly 420 425 430 TyrLys Met Thr Ser Tyr Gln Glu Leu Met Ser Phe Ser Ser Pro Leu 435 440 445Phe Gly Phe Ser Phe Ala Asp Val Pro Val Asp Lys Ala Ala Asn Tyr 450 455460 Ser Cys Glu Asp Ala Asp Ile Thr Tyr Arg Leu Tyr Lys Ile Leu Ser 465470 475 480 Met Lys Leu His Glu Ala Glu Leu Glu Asn Val Phe Tyr Arg IleGlu 485 490 495 Met Pro Leu Val Asn Val Leu Ala Arg Met Glu Leu Asn GlyVal Tyr 500 505 510 Val Asp Thr Glu Phe Leu Lys Lys Leu Ser Glu Glu TyrGly Lys Lys 515 520 525 Leu Glu Glu Leu Ala Glu Lys Ile Tyr Gln Ile AlaGly Glu Pro Phe 530 535 540 Asn Ile Asn Ser Pro Lys Gln Val Ser Lys IleLeu Phe Glu Lys Leu 545 550 555 560 Gly Ile Lys Pro Arg Gly Lys Thr ThrLys Thr Gly Glu Tyr Ser Thr 565 570 575 Arg Ile Glu Val Leu Glu Glu IleAla Asn Glu His Glu Ile Val Pro 580 585 590 Leu Ile Leu Glu Tyr Arg LysIle Gln Lys Leu Lys Ser Thr Tyr Ile 595 600 605 Asp Thr Leu Pro Lys LeuVal Asn Pro Lys Thr Gly Arg Ile His Ala 610 615 620 Ser Phe His Gln ThrGly Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp 625 630 635 640 Pro Asn LeuGln Asn Leu Pro Thr Lys Ser Glu Glu Gly Lys Glu Ile 645 650 655 Arg LysAla Ile Val Pro Gln Asp Pro Asp Trp Trp Ile Val Ser Ala 660 665 670 AspTyr Ser Gln Ile Glu Leu Arg Ile Leu Ala His Leu Ser Gly Asp 675 680 685Glu Asn Leu Val Lys Ala Phe Glu Glu Gly Ile Asp Val His Thr Leu 690 695700 Thr Ala Ser Arg Ile Tyr Asn Val Lys Pro Glu Glu Val Asn Glu Glu 705710 715 720 Met Arg Arg Val Gly Lys Met Val Asn Phe Ser Ile Ile Tyr GlyVal 725 730 735 Thr Pro Tyr Gly Leu Ser Val Arg Leu Gly Ile Pro Val LysGlu Ala 740 745 750 Glu Lys Met Ile Ile Ser Tyr Phe Thr Leu Tyr Pro LysVal Arg Ser 755 760 765 Tyr Ile Gln Gln Val Val Ala Glu Ala Lys Glu LysGly Tyr Val Arg 770 775 780 Thr Leu Phe Gly Arg Lys Arg Asp Ile Pro GlnLeu Met Ala Arg Asp 785 790 795 800 Lys Asn Thr Gln Ser Glu Gly Glu ArgIle Ala Ile Asn Thr Pro Ile 805 810 815 Gln Gly Thr Ala Ala Asp Ile IleLys Leu Ala Met Ile Asp Ile Asp 820 825 830 Glu Glu Leu Arg Lys Arg AsnMet Lys Ser Arg Met Ile Ile Gln Val 835 840 845 His Asp Glu Leu Val PheGlu Val Pro Asp Glu Glu Lys Glu Glu Leu 850 855 860 Val Asp Leu Val LysAsn Lys Met Thr Asn Val Val Lys Leu Ser Val 865 870 875 880 Pro Leu GluVal Asp Ile Ser Ile Gly Lys Ser Trp Ser 885 890 893 amino acids aminoacid not relevant not relevant protein 8 Met Ala Arg Leu Phe Leu Phe AspGly Thr Ala Leu Ala Tyr Arg Ala 1 5 10 15 Tyr Tyr Ala Leu Asp Arg SerLeu Ser Thr Ser Thr Gly Ile Pro Thr 20 25 30 Asn Ala Val Tyr Gly Val AlaArg Met Leu Val Lys Phe Ile Lys Glu 35 40 45 His Ile Ile Pro Glu Lys AspTyr Ala Ala Val Ala Phe Asp Lys Lys 50 55 60 Ala Ala Thr Phe Arg His LysLeu Leu Val Ser Asp Lys Ala Gln Arg 65 70 75 80 Pro Lys Thr Pro Ala LeuLeu Val Gln Gln Leu Pro Tyr Ile Lys Arg 85 90 95 Leu Ile Glu Ala Leu GlyPhe Lys Val Leu Glu Leu Glu Gly Tyr Glu 100 105 110 Ala Asp Asp Ile IleAla Thr Leu Ala Val Arg Ala Ala Arg Phe Leu 115 120 125 Met Arg Phe SerLeu Ile Thr Gly Asp Lys Asp Met Leu Gln Leu Val 130 135 140 Asn Glu LysIle Lys Val Trp Arg Ile Val Lys Gly Ile Ser Asp Leu 145 150 155 160 GluLeu Tyr Asp Ser Lys Lys Val Lys Glu Arg Tyr Gly Val Glu Pro 165 170 175His Gln Ile Pro Asp Leu Leu Ala Leu Thr Gly Asp Asp Ile Asp Asn 180 185190 Ile Pro Asp Val Thr Gly Ile Gly Glu Lys Thr Ala Val Gln Leu Leu 195200 205 Gly Lys Tyr Arg Asn Leu Glu Tyr Ile Leu Glu His Ala Arg Glu Leu210 215 220 Pro Gln Arg Val Arg Lys Ala Leu Leu Arg Asp Arg Glu Val AlaIle 225 230 235 240 Leu Ser Lys Lys Leu Ala Thr Leu Val Thr Asn Ala ProVal Glu Val 245 250 255 Asp Trp Glu Glu Met Lys Tyr Arg Gly Tyr Asp LysArg Lys Leu Leu 260 265 270 Pro Ile Leu Lys Glu Leu Glu Phe Ala Ser IleMet Lys Glu Leu Gln 275 280 285 Leu Tyr Glu Glu Ala Glu Pro Thr Gly TyrGlu Ile Val Lys Asp His 290 295 300 Lys Thr Phe Glu Asp Leu Ile Glu LysLeu Lys Glu Val Pro Ser Phe 305 310 315 320 Ala Leu Ala Leu Glu Thr SerSer Leu Asp Pro Phe Asn Cys Glu Ile 325 330 335 Val Gly Ile Ser Val SerPhe Lys Pro Lys Thr Ala Tyr Tyr Ile Pro 340 345 350 Leu His His Arg AsnAla Gln Asn Leu Asp Glu Thr Leu Val Leu Ser 355 360 365 Lys Leu Lys GluIle Leu Glu Asp Pro Ser Ser Lys Ile Val Gly Gln 370 375 380 Asn Leu LysTyr Asp Tyr Lys Val Leu Met Val Lys Gly Ile Ser Pro 385 390 395 400 ValTyr Pro His Phe Asp Thr Met Ile Ala Ala Tyr Leu Leu Glu Pro 405 410 415Asn Glu Lys Lys Phe Asn Leu Glu Asp Leu Ser Leu Lys Phe Leu Gly 420 425430 Tyr Lys Met Thr Ser Tyr Gln Glu Leu Met Ser Phe Ser Ser Pro Leu 435440 445 Phe Gly Phe Ser Phe Ala Asp Val Pro Val Asp Lys Ala Ala Asn Tyr450 455 460 Ser Cys Glu Asp Ala Asp Ile Thr Tyr Arg Leu Tyr Lys Ile LeuSer 465 470 475 480 Met Lys Leu His Glu Ala Glu Leu Glu Asn Val Phe TyrArg Ile Glu 485 490 495 Met Pro Leu Val Asn Val Leu Ala Arg Met Glu LeuAsn Gly Val Tyr 500 505 510 Val Asp Thr Glu Phe Leu Lys Lys Leu Ser GluGlu Tyr Gly Lys Lys 515 520 525 Leu Glu Glu Leu Ala Glu Lys Ile Tyr GlnIle Ala Gly Glu Pro Phe 530 535 540 Asn Ile Asn Ser Pro Lys Gln Val SerLys Ile Leu Phe Glu Lys Leu 545 550 555 560 Gly Ile Lys Pro Arg Gly LysThr Thr Lys Thr Gly Glu Tyr Ser Thr 565 570 575 Arg Ile Glu Val Leu GluGlu Ile Ala Asn Glu His Glu Ile Val Pro 580 585 590 Leu Ile Leu Glu TyrArg Lys Ile Gln Lys Leu Lys Ser Thr Tyr Ile 595 600 605 Asp Thr Leu ProLys Leu Val Asn Pro Lys Thr Gly Arg Ile His Ala 610 615 620 Ser Phe HisGln Thr Gly Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp 625 630 635 640 ProAsn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly Lys Glu Ile 645 650 655Arg Lys Ala Ile Val Pro Gln Asp Pro Asp Trp Trp Ile Val Ser Ala 660 665670 Asp Tyr Ser Gln Ile Glu Leu Arg Ile Leu Ala His Leu Ser Gly Asp 675680 685 Glu Asn Leu Val Lys Ala Phe Glu Glu Gly Ile Asp Val His Thr Leu690 695 700 Thr Ala Ser Arg Ile Tyr Asn Val Lys Pro Glu Glu Val Asn GluGlu 705 710 715 720 Met Arg Arg Val Gly Lys Met Val Asn Phe Ser Ile IleTyr Gly Val 725 730 735 Thr Pro Tyr Gly Leu Ser Val Arg Leu Gly Ile ProVal Lys Glu Ala 740 745 750 Glu Lys Met Ile Ile Ser Tyr Phe Thr Leu TyrPro Lys Val Arg Ser 755 760 765 Tyr Ile Gln Gln Val Val Ala Glu Ala LysGlu Lys Gly Tyr Val Arg 770 775 780 Thr Leu Phe Gly Arg Lys Arg Asp IlePro Gln Leu Met Ala Arg Asp 785 790 795 800 Lys Asn Thr Gln Ser Glu GlyGlu Arg Ile Ala Ile Asn Thr Pro Ile 805 810 815 Gln Gly Thr Ala Ala AspIle Ile Lys Leu Ala Met Ile Asp Ile Asp 820 825 830 Glu Glu Leu Arg LysArg Asn Met Lys Ser Arg Met Ile Ile Gln Val 835 840 845 His Asp Glu LeuVal Phe Glu Val Pro Asp Glu Glu Lys Glu Glu Leu 850 855 860 Val Asp LeuVal Lys Asn Lys Met Thr Asn Val Val Lys Leu Ser Val 865 870 875 880 ProLeu Glu Val Asp Ile Ser Ile Gly Lys Ser Trp Ser 885 890 893 amino acidsamino acid not relevant not relevant protein 9 Met Ala Arg Leu Phe LeuPhe Asp Gly Thr Ala Leu Ala Tyr Arg Ala 1 5 10 15 Tyr Tyr Ala Leu AspArg Ser Leu Ser Thr Ser Thr Gly Ile Pro Thr 20 25 30 Asn Ala Val Tyr AspVal Ala Arg Met Leu Val Lys Phe Ile Lys Glu 35 40 45 His Ile Ile Pro GluLys Asp Tyr Ala Ala Val Ala Phe Asp Lys Lys 50 55 60 Ala Ala Thr Phe ArgHis Lys Leu Leu Val Ser Asp Lys Ala Gln Arg 65 70 75 80 Pro Lys Thr ProAla Leu Leu Val Gln Gln Leu Pro Tyr Ile Lys Arg 85 90 95 Leu Ile Glu AlaLeu Gly Phe Lys Val Leu Glu Leu Glu Gly Tyr Glu 100 105 110 Ala Asp AspIle Ile Ala Thr Leu Ala Val Arg Ala Ala Arg Phe Leu 115 120 125 Met ArgPhe Ser Leu Ile Thr Gly Asp Lys Asp Met Leu Gln Leu Val 130 135 140 AsnGlu Lys Ile Lys Val Trp Arg Ile Val Lys Gly Ile Ser Asp Leu 145 150 155160 Glu Leu Tyr Asp Ser Lys Lys Val Lys Glu Arg Tyr Gly Val Glu Pro 165170 175 His Gln Ile Pro Asp Leu Leu Ala Leu Thr Gly Asp Asp Ile Asp Asn180 185 190 Ile Pro Gly Val Thr Gly Ile Gly Glu Lys Thr Ala Val Gln LeuLeu 195 200 205 Gly Lys Tyr Arg Asn Leu Glu Tyr Ile Leu Glu His Ala ArgGlu Leu 210 215 220 Pro Gln Arg Val Arg Lys Ala Leu Leu Arg Asp Arg GluVal Ala Ile 225 230 235 240 Leu Ser Lys Lys Leu Ala Thr Leu Val Thr AsnAla Pro Val Glu Val 245 250 255 Asp Trp Glu Glu Met Lys Tyr Arg Gly TyrAsp Lys Arg Lys Leu Leu 260 265 270 Pro Ile Leu Lys Glu Leu Glu Phe AlaSer Ile Met Lys Glu Leu Gln 275 280 285 Leu Tyr Glu Glu Ala Glu Pro ThrGly Tyr Glu Ile Val Lys Asp His 290 295 300 Lys Thr Phe Glu Asp Leu IleGlu Lys Leu Lys Glu Val Pro Ser Phe 305 310 315 320 Ala Leu Ala Leu GluThr Ser Ser Leu Asp Pro Phe Asn Cys Glu Ile 325 330 335 Val Gly Ile SerVal Ser Phe Lys Pro Lys Thr Ala Tyr Tyr Ile Pro 340 345 350 Leu His HisArg Asn Ala Gln Asn Leu Asp Glu Thr Leu Val Leu Ser 355 360 365 Lys LeuLys Glu Ile Leu Glu Asp Pro Ser Ser Lys Ile Val Gly Gln 370 375 380 AsnLeu Lys Tyr Asp Tyr Lys Val Leu Met Val Lys Gly Ile Ser Pro 385 390 395400 Val Tyr Pro His Phe Asp Thr Met Ile Ala Ala Tyr Leu Leu Glu Pro 405410 415 Asn Glu Lys Lys Phe Asn Leu Glu Asp Leu Ser Leu Lys Phe Leu Gly420 425 430 Tyr Lys Met Thr Ser Tyr Gln Glu Leu Met Ser Phe Ser Ser ProLeu 435 440 445 Phe Gly Phe Ser Phe Ala Asp Val Pro Val Asp Lys Ala AlaAsn Tyr 450 455 460 Ser Cys Glu Asp Ala Asp Ile Thr Tyr Arg Leu Tyr LysIle Leu Ser 465 470 475 480 Met Lys Leu His Glu Ala Glu Leu Glu Asn ValPhe Tyr Arg Ile Glu 485 490 495 Met Pro Leu Val Asn Val Leu Ala Arg MetGlu Leu Asn Gly Val Tyr 500 505 510 Val Asp Thr Glu Phe Leu Lys Lys LeuSer Glu Glu Tyr Gly Lys Lys 515 520 525 Leu Glu Glu Leu Ala Glu Lys IleTyr Gln Ile Ala Gly Glu Pro Phe 530 535 540 Asn Ile Asn Ser Pro Lys GlnVal Ser Lys Ile Leu Phe Glu Lys Leu 545 550 555 560 Gly Ile Lys Pro ArgGly Lys Thr Thr Lys Thr Gly Glu Tyr Ser Thr 565 570 575 Arg Ile Glu ValLeu Glu Glu Ile Ala Asn Glu His Glu Ile Val Pro 580 585 590 Leu Ile LeuGlu Tyr Arg Lys Ile Gln Lys Leu Lys Ser Thr Tyr Ile 595 600 605 Asp ThrLeu Pro Lys Leu Val Asn Pro Lys Thr Gly Arg Ile His Ala 610 615 620 SerPhe His Gln Thr Gly Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp 625 630 635640 Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu Glu Gly Lys Glu Ile 645650 655 Arg Lys Ala Ile Val Pro Gln Asp Pro Asp Trp Trp Ile Val Ser Ala660 665 670 Asp Tyr Ser Gln Ile Glu Leu Arg Ile Leu Ala His Leu Ser GlyAsp 675 680 685 Glu Asn Leu Val Lys Ala Phe Glu Glu Gly Ile Asp Val HisThr Leu 690 695 700 Thr Ala Ser Arg Ile Tyr Asn Val Lys Pro Glu Glu ValAsn Glu Glu 705 710 715 720 Met Arg Arg Val Gly Lys Met Val Asn Phe SerIle Ile Tyr Gly Val 725 730 735 Thr Pro Tyr Gly Leu Ser Val Arg Leu GlyIle Pro Val Lys Glu Ala 740 745 750 Glu Lys Met Ile Ile Ser Tyr Phe ThrLeu Tyr Pro Lys Val Arg Ser 755 760 765 Tyr Ile Gln Gln Val Val Ala GluAla Lys Glu Lys Gly Tyr Val Arg 770 775 780 Thr Leu Phe Gly Arg Lys ArgAsp Ile Pro Gln Leu Met Ala Arg Asp 785 790 795 800 Lys Asn Thr Gln SerGlu Gly Glu Arg Ile Ala Ile Asn Thr Pro Ile 805 810 815 Gln Gly Thr AlaAla Asp Ile Ile Lys Leu Ala Met Ile Asp Ile Asp 820 825 830 Glu Glu LeuArg Lys Arg Asn Met Lys Ser Arg Met Ile Ile Gln Val 835 840 845 His AspGlu Leu Val Phe Glu Val Pro Asp Glu Glu Lys Glu Glu Leu 850 855 860 ValAsp Leu Val Lys Asn Lys Met Thr Asn Val Val Lys Leu Ser Val 865 870 875880 Pro Leu Glu Val Asp Ile Ser Ile Gly Lys Ser Trp Ser 885 890 610amino acids amino acid not relevant not relevant protein 10 Met Lys GluLeu Gln Leu Tyr Glu Glu Ala Glu Pro Thr Gly Tyr Glu 1 5 10 15 Ile ValLys Asp His Lys Thr Phe Glu Asp Leu Ile Glu Lys Leu Lys 20 25 30 Glu ValPro Ser Phe Ala Leu Asp Leu Glu Thr Ser Ser Leu Asp Pro 35 40 45 Phe AsnCys Glu Ile Val Gly Ile Ser Val Ser Phe Lys Pro Lys Thr 50 55 60 Ala TyrTyr Ile Pro Leu His His Arg Asn Ala Gln Asn Leu Asp Glu 65 70 75 80 ThrLeu Val Leu Ser Lys Leu Lys Glu Ile Leu Glu Asp Pro Ser Ser 85 90 95 LysIle Val Gly Gln Asn Leu Lys Tyr Asp Tyr Lys Val Leu Met Val 100 105 110Lys Gly Ile Ser Pro Val Tyr Pro His Phe Asp Thr Met Ile Ala Ala 115 120125 Tyr Leu Leu Glu Pro Asn Glu Lys Lys Phe Asn Leu Glu Asp Leu Ser 130135 140 Leu Lys Phe Leu Gly Tyr Lys Met Thr Ser Tyr Gln Glu Leu Met Ser145 150 155 160 Phe Ser Ser Pro Leu Phe Gly Phe Ser Phe Ala Asp Val ProVal Asp 165 170 175 Lys Ala Ala Asn Tyr Ser Cys Glu Asp Ala Asp Ile ThrTyr Arg Leu 180 185 190 Tyr Lys Ile Leu Ser Met Lys Leu His Glu Ala GluLeu Glu Asn Val 195 200 205 Phe Tyr Arg Ile Glu Met Pro Leu Val Asn ValLeu Ala Arg Met Glu 210 215 220 Leu Asn Gly Val Tyr Val Asp Thr Glu PheLeu Lys Lys Leu Ser Glu 225 230 235 240 Glu Tyr Gly Lys Lys Leu Glu GluLeu Ala Glu Lys Ile Tyr Gln Ile 245 250 255 Ala Gly Glu Pro Phe Asn IleAsn Ser Pro Lys Gln Val Ser Lys Ile 260 265 270 Leu Phe Glu Lys Leu GlyIle Lys Pro Arg Gly Lys Thr Thr Lys Thr 275 280 285 Gly Glu Tyr Ser ThrArg Ile Glu Val Leu Glu Glu Ile Ala Asn Glu 290 295 300 His Glu Ile ValPro Leu Ile Leu Glu Tyr Arg Lys Ile Gln Lys Leu 305 310 315 320 Lys SerThr Tyr Ile Asp Thr Leu Pro Lys Leu Val Asn Pro Lys Thr 325 330 335 GlyArg Ile His Ala Ser Phe His Gln Thr Gly Thr Ala Thr Gly Arg 340 345 350Leu Ser Ser Ser Asp Pro Asn Leu Gln Asn Leu Pro Thr Lys Ser Glu 355 360365 Glu Gly Lys Glu Ile Arg Lys Ala Ile Val Pro Gln Asp Pro Asp Trp 370375 380 Trp Ile Val Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile Leu Ala385 390 395 400 His Leu Ser Gly Asp Glu Asn Leu Val Lys Ala Phe Glu GluGly Ile 405 410 415 Asp Val His Thr Leu Thr Ala Ser Arg Ile Tyr Asn ValLys Pro Glu 420 425 430 Glu Val Asn Glu Glu Met Arg Arg Val Gly Lys MetVal Asn Phe Ser 435 440 445 Ile Ile Tyr Gly Val Thr Pro Tyr Gly Leu SerVal Arg Leu Asn Ile 450 455 460 Pro Val Lys Glu Ala Glu Lys Met Ile IleSer Tyr Phe Thr Leu Tyr 465 470 475 480 Pro Lys Val Arg Ser Tyr Ile GlnGln Val Val Ala Glu Ala Lys Glu 485 490 495 Lys Gly Tyr Val Arg Thr LeuPhe Gly Arg Lys Arg Asp Ile Pro Gln 500 505 510 Leu Met Ala Arg Asp LysAsn Thr Gln Ser Glu Gly Glu Arg Ile Ala 515 520 525 Ile Asn Thr Pro IleGln Gly Thr Ala Ala Asp Ile Ile Lys Leu Ala 530 535 540 Met Ile Asp IleAsp Glu Glu Leu Arg Lys Arg Asn Met Lys Ser Arg 545 550 555 560 Met IleIle Gln Val His Asp Glu Leu Val Phe Glu Val Pro Asp Glu 565 570 575 GluLys Glu Glu Leu Val Asp Leu Val Lys Asn Lys Met Thr Asn Val 580 585 590Val Lys Leu Ser Val Pro Leu Glu Val Asp Ile Ser Ile Gly Lys Ser 595 600605 Trp Ser 610 14 amino acids amino acid not relevant linear peptideModified-site 1..14 /note= “′Xaa′ is any amino acid” 11 Arg Xaa Xaa XaaLys Xaa Xaa Xaa Phe Xaa Xaa Xaa Tyr Xaa 1 5 10 14 amino acids amino acidnot relevant linear peptide 12 Arg Arg Ser Ala Lys Ala Ile Asn Phe GlyLeu Ile Tyr Gly 1 5 10 14 amino acids amino acid not relevant linearpeptide 13 Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 1 510 14 amino acids amino acid not relevant linear peptide 14 Arg Asp AsnAla Lys Thr Phe Ile Tyr Gly Phe Leu Tyr Gly 1 5 10 14 amino acids aminoacid not relevant linear peptide 15 Arg Arg Val Gly Lys Met Val Asn PheSer Ile Ile Tyr Gly 1 5 10 14 amino acids amino acid not relevant linearpeptide 16 Arg Gln Ala Ala Lys Ala Ile Thr Phe Gly Ile Leu Tyr Gly 1 510 14 amino acids amino acid not relevant linear peptide 17 Arg Arg AlaGly Lys Met Val Asn Phe Ser Ile Ile Tyr Gly 1 5 10 11 amino acids aminoacid not relevant linear peptide 18 Pro Ser Phe Ala Leu Asp Leu Glu ThrSer Ser 1 5 10 11 amino acids amino acid not relevant linear peptide 19Pro Val Phe Ala Phe Asp Thr Glu Thr Asp Ser 1 5 10 11 amino acids aminoacid not relevant linear peptide 20 Gly Pro Val Ala Phe Asp Ser Glu ThrSer Ala 1 5 10 10 amino acids amino acid not relevant linear peptide 21Met Ile Val Ser Asp Ile Glu Ala Asn Ala 1 5 10 26 base pairs nucleicacid both both cDNA 22 GACGTTTCAA GCGCTAGGGC AAAAGA 26 31 base pairsnucleic acid both both cDNA 23 GTATATTATA GAGTAGTTAA CCATCTTTCC A 31 6amino acids amino acid not relevant linear 24 Phe Leu Phe Asp Gly Thr 15 6 amino acids amino acid not relevant linear peptide 25 Leu Leu ValAsp Gly His 1 5 10 amino acids amino acid not relevant linear peptide 26Ser Leu Ile Thr Gly Asp Lys Asp Met Leu 1 5 10 10 amino acids amino acidnot relevant linear peptide 27 Arg Ile Leu Thr Ala Asp Lys Asp Leu Tyr 15 10 35 base pairs nucleic acid both both cDNA 28 GTAGGCCAGG GCTGTGCCGGCAAAGAGAAA TAGTC 35 35 base pairs nucleic acid both both cDNA 29GAAGCATATC CTTGGCGCCG GTTATTATGA AAATC 35 30 base pairs nucleic acidboth both cDNA 30 CACCAGACGG GTACCGCCAC TGGCAGGTTG 30 48 base pairsnucleic acid both both cDNA 31 TATAGAGTAG TTAACCATCT TTCCAACCCGTTTCATTTCT TCGAACAC 48 48 base pairs nucleic acid both both cDNA 32TATAGAGTAG TTAACCATCT TTCCAACCCG TTGCATTTCT TCGAACAC 48 48 base pairsnucleic acid both both cDNA 33 TATAGAGTAG TTAACCATCT TTCCAACCCGGTTCATTTCT TCGAACAC 48 48 base pairs nucleic acid both both cDNA 34TATAGAGTAG TTAACCATCT TTCCAACCCG ATGCATTTCT TCGAACAC 48 29 base pairsnucleic acid both both cDNA 35 AAGATGGTTA ACGCGTCTAT AATATACGG 29 23base pairs nucleic acid both both cDNA 36 CAAGAGGCAC AGAGAGTTTC ACC 2330 base pairs nucleic acid both both cDNA 37 GTATATTATA GAGGAGTTAACCATCTTTCC 30 29 base pairs nucleic acid both both cDNA 38 AAGATGGTTAACTTCTCTAT AATATACGG 29 48 base pairs nucleic acid both both cDNA 39TATAGAGTAG TTAACCATCT TTCCAACCCG GTACATGTCT TCGTTCAC 48 48 base pairsnucleic acid both both cDNA 40 TATAGAGTAG TTAACCATCT TTCCAACCCGCAACATGTCT TCGTTCAC 48 27 base pairs nucleic acid both both cDNA 41CTTGGCCGCC CGATGCATCA GGGGGTC 27 30 base pairs nucleic acid both bothcDNA 42 CTTGGCCGCC CGCTTCATGA GGGGGTCCAC 30 27 base pairs nucleic acidboth both cDNA 43 CTTGGCCGCC CTGTACATCA GGGGGTC 27 30 base pairs nucleicacid both both cDNA 44 GTATATTATA GAGGTGTTAA CCATCTTTCC 30 34 base pairsnucleic acid both both cDNA 45 GGGAGACCGG AATTCTCCTT CATTAATTCC TATA 3449 base pairs nucleic acid both both cDNA 46 TGGAGACCCT GGAACTATAGGAATTAATGA AGGAGAATTC CGGTCTCCC 49 20 base pairs nucleic acid both bothcDNA 47 GTATTTTGGT ATGCTTGTGC 20 22 base pairs nucleic acid both bothcDNA 48 CTATTTTGGA ATATATGTGC CT 22 20 base pairs nucleic acid both bothcDNA 49 ACGAACATTC TACAAGTTAC 20 20 base pairs nucleic acid both bothcDNA 50 TTTCAGAGAA ACTGACCTGT 20 21 base pairs nucleic acid both bothcDNA 51 GATAAATGCC AAACATGTTG T 21 20 base pairs nucleic acid both bothcDNA 52 TGCTCTCAGG ATTTCCTCCA 20 20 base pairs nucleic acid both bothcDNA 53 AGCTTGAGAC CTCTGTGTCC 20 22 base pairs nucleic acid both bothcDNA 54 ATTCAGAAGA AACAGTGATG GT 22 23 base pairs nucleic acid both bothcDNA 55 TTGGAGTCGC AAGCTGAACT AGC 23 23 base pairs nucleic acid bothboth cDNA 56 GCCTGAGTGA CAGAGTGAGA ACC 23 24 base pairs nucleic acidboth both cDNA 57 CCCACTAGGT TGTAAGCTCC ATGA 24 24 base pairs nucleicacid both both cDNA 58 TACTATGTGC CAGGCTCTGT CCTA 24 20 base pairsnucleic acid both both cDNA 59 ACTCATGAAG GTGACAGTTC 20 20 base pairsnucleic acid both both cDNA 60 GTGTTGTTGA CCTATTGCAT 20 20 base pairsnucleic acid both both cDNA 61 ATCTCTGTTC CCTCCCTGTT 20 20 base pairsnucleic acid both both cDNA 62 CTTATTGGCC TTGAAGGTAG 20 23 base pairsnucleic acid both both cDNA 63 AGCCCGTGTT GGAACCATGA CTG 23 23 basepairs nucleic acid both both cDNA 64 TACATAGCGA GACTCCATCT CCC 23 20base pairs nucleic acid both both cDNA 65 TTTATGCGAG CGTATGGATA 20 20base pairs nucleic acid both both cDNA 66 CACCACCATT GATCTGGAAG 20 16base pairs nucleic acid both both cDNA 67 CCAACCACAC TGGGAA 16 16 basepairs nucleic acid both both cDNA 68 AACAGTTGCC CACGGT 16 20 base pairsnucleic acid both both cDNA 69 CATGAAATGC TGACTGGGTA 20 20 base pairsnucleic acid both both cDNA 70 TCAATTTATG TGCAGCCAAT 20 20 base pairsnucleic acid both both cDNA 71 CATAGCGAGA CTCCATCTCC 20 20 base pairsnucleic acid both both cDNA 72 GGGAGAGGGC AAAGATCGAT 20 22 base pairsnucleic acid both both cDNA 73 AACACTAGTG ACATTATTTT CA 22 20 base pairsnucleic acid both both cDNA 74 AGCTAGGCCT GAAGGCTTCT 20 24 base pairsnucleic acid both both cDNA 75 CCCTAGTGGA TGATAAGAAT AATC 24 30 basepairs nucleic acid both both cDNA 76 GGACAGATGA TAAATACATA GGATGGATGG 3020 base pairs nucleic acid both both cDNA 77 TTCTCTTACA ACACTGCCCC 20 20base pairs nucleic acid both both cDNA 78 ATTTGGATGG CTTGACAGAG 20 21base pairs nucleic acid both both cDNA 79 ACATTCTAAG ACTTTCCCAA T 21 20base pairs nucleic acid both both cDNA 80 AGAGCATGCA CCCTGAATTG 20 20base pairs nucleic acid both both cDNA 81 AAGAACCATG CGATACGACT 20 20base pairs nucleic acid both both cDNA 82 CATTCCTAGA TGGGTAAAGC 20 18base pairs nucleic acid both both cDNA 83 GCTTAGTCAT ACGAGCGG 18 18 basepairs nucleic acid both both cDNA 84 TCCACAGCCA TGTAAACC 18 16 basepairs nucleic acid both both cDNA 85 CCCCGGAGCA AGTTCA 16 18 base pairsnucleic acid both both cDNA 86 CAGCCCAAAG CCAGATTA 18 22 base pairsnucleic acid both both cDNA 87 ATATGTGAGT CAATTCCCCA AG 22 22 base pairsnucleic acid both both cDNA 88 TGTATTAGTC AATGTTCTCC AG 22 19 base pairsnucleic acid both both cDNA 89 CAGCTGCCCT AGTCAGCAC 19 20 base pairsnucleic acid both both cDNA 90 GCTTCCGAGT GCAGGTCACA 20 21 base pairsnucleic acid both both cDNA 91 ATTCTGGGCG CACAAGAGTG A 21 20 base pairsnucleic acid both both cDNA 92 ACATCTCCCC TACCGCTATA 20 33 base pairsnucleic acid both both cDNA 93 GAAGTTCACC ATCCGGCCGA CCCGTCGCAT TTC 33

What is claimed is:
 1. A method of identifying, analyzing or typing apolymorphic DNA fragment in a sample of DNA, said method comprisingcontacting said sample of DNA with one or more DNA polymerasessubstantially reduced in the ability to add one or more non-templatednucleotides to the 3′ terminus of a DNA molecule, amplifying saidpolymorphic DNA fragment within said sample and analyzing said amplifiedpolymorphic DNA fragment.
 2. A method of producing amplified copies of apolymorphic DNA fragment which comprise substantially no non-templated3′ terminal nucleotides, said method comprising contacting a DNA samplewith one or more DNA polymerases substantially reduced in the ability toadd one or more non-templated nucleotides to the 3′ terminus of a DNAmolecule and amplifying said polymorphic DNA fragment within said DNAsample.
 3. A method of cloning a DNA molecule comprising contacting saidDNA molecule with one or more DNA polymerases substantially reduced inthe ability to add one or more non-templated nucleotides to the 3′terminus of a DNA molecule, amplifying said DNA molecule and insertingsaid DNA molecule into a vector.
 4. The method of claim 3, wherein saidvector is blunt-ended.
 5. The method of claim 1, wherein saidpolymorphic DNA fragment is selected from the group of polymorphic DNAfragments comprising a minisatellite DNA fragment, a microsatellite DNAfragment and a STR DNA fragment.
 6. The method of claim 1, wherein saidpolymerases are thermostable DNA polymerases.
 7. The method of claim 6,wherein said thermostable DNA polymerases are Thermotoga DNA polymerasesand mutants or derivatives thereof.
 8. The method of claim 7, whereinsaid DNA polymerase is a Tne or Tma DNA polymerase.
 9. The method ofclaim 1, wherein said DNA polymerases are substantially reduced in 3′-5′exonuclease activity.
 10. The method of claim 1, wherein said DNApolymerases are substantially reduced in 5′-3′ exonuclease activity. 11.The method of claim 9, wherein said DNA polymerases are substantiallyreduced in 5′-3′ exonuclease activity.
 12. The method of claim 1,wherein said DNA polymerases contain one or more modifications ormutations which reduce the ability of the polymerase to add one or morenon-templated 3′ nucleotides to a synthesized nucleic acid molecule. 13.The method of claim 12, wherein said DNA polymerases are substantiallyreduced in at least one activity selected from the group consisting of:(a) 3′-5′ exonuclease activity; and (b) 5′-3′ exonuclease activity. 14.The method of claim 13, wherein said polymerases have substantiallyreduced 3′-5′ exonuclease and 5′-3′ exonuclease activity.
 15. The methodof claim 13, wherein said polymerase is substantially reduced in 3′-5′exonuclease activity.
 16. The method of claim 12, wherein saidpolymerases comprise one or more mutations or modifications in theO-helix of said polymerase.
 17. The method of claim 16, wherein saidO-helix is defined as RXXXKXXXFXXXYX (SEQ ID NO:11), wherein X is anyamino acid.
 18. The method of claim 17, wherein said mutation ormodification is at position R (Arg) and/or F (Phe) and/or K (Lys) ofsaid O-helix or combinations thereof.
 20. The method of claim 16,wherein said mutation or modification is an amino acid substitution atposition R and/or F and/or K of said O-helix or combinations thereof.19. The method of claim 1, wherein said polymerase is selected from thegroup consisting of: Tne N′Δ219, D323A; Tne N′Δ283, D323A; Tne N′Δ284,D323A; Tne N′Δ193, D323A; Tne D137A, D323A; Tne D8A, D323A; Tne G195D,D323A; Tne G37D, D323A, Tne N′Δ283; Tne D137A, D323A, R722K; Tne D137A,D323A, R722Y; Tne D137A, D323A, R722L; Tne D137A, D323A, R722H; TneD137A, D323A, R722Q; Tne D137A, D323A, F730Y; Tne D137A, D323A, K726R;Tne D137A, D323A, K726H; Tne D137A, D323A, R722K, F730Y; Tne D137A,D323A, R722K, K726R; Tne D137A, D323A, R722K, K726H; Tne D137A, D323A,R722H, F730Y; Tne D137A, D323A, R722H, K726R; Tne D137A, D323A, R722H,K726H; Tne D137A, D323A, R722Q, F730Y; Tne D137A, D323A, R722Q, K726R;Tne D137A, D323A, R722Q, K726H; Tne D137A, D323A, R722N, F730Y; TneD137A, D323A, R722N, K726R; Tne D137A, D323A, R722N, K726H; Tne D137A,D323A, F730S; Tne N′Δ283, D323A, R722K/H/Q/N/Y/L; Tne N′Δ219, D323A,R722K; Tne N′Δ219, D323A, F730Y; Tne N′Δ219, D323A, K726R; Tne N′Δ219,D323A, K726H; Tne D137A, D323A, F730S, R722K/Y/Q/N/H/L, K726R/H; TneD137A, D323A, F730T, R722K/Y/Q/N/H/L, K726R/H; Tne D137A, D323A, F730T;Tne F730S; Tne F730A; Tne K726R; Tne K726H; and Tne D137A, D323A, R722N.21. A method of determining the relationship between a first individualand a second individual, said method comprising comparing a populationof amplified DNA molecules in a sample of DNA from said first individualto that of said second individual prepared according to the method ofclaim
 1. 22. The method of claim 21, wherein said sample of DNA fromsaid first individual is a known sample and said sample of DNA from saidsecond individual is an unknown sample.
 23. A kit for theidentification, analysis or typing of a polymorphic DNA fragment, saidkit comprising one or more DNA polymerases substantially reduced in theability to add one or more non-templated nucleotides to the 3′ terminusof a DNA molecule.
 24. The kit of claim 23, said kit further comprisingone or more components selected from the group consisting of one or moreDNA primers, one or more deoxynucleoside triphosphates, and a buffersuitable for use in the identification, analysis or typing of apolymorphic DNA fragment.
 25. The kit of claim 23, wherein saidpolymerases are thermostable DNA polymerases.
 26. The kit of claim 25,wherein thermostable DNA polymerases are Thermotoga DNA polymerases. 27.The kit of claim 23, wherein said DNA polymerase is substantiallyreduced in 3′-5′ exonuclease activity.
 28. The kit of claim 23, whereinsaid DNA polymerase is substantially reduced in 5′-3′ exonucleaseactivity.
 29. The kit of claim 23, wherein said DNA polymerases compriseone or more modifications or mutations which reduce the ability of thepolymerase to add one or more non-templated 3′ nucleotides to asynthesized nucleic acid molecule.
 30. The kit of claim 29, wherein saidpolymerases comprise one or more mutations in the O-helix of saidpolymerase.
 31. The kit of claim 30, wherein said O-helix is defined asRXXXKXXXFXXXYX (SEQ ID NO:11), wherein X is any amino acid.
 32. The kitof claim 31, wherein said mutation or modification is at position R(Arg) and/or F (Phe) and/or K (Lys) of said O-helix or combinationsthereof.
 33. The method of claim 31, wherein said mutation ormodification is an amino acid substitution at position R and/or F and/orK of said O-helix or combinations thereof.
 34. A polymerase which hasbeen modified or mutated to reduce, substantially reduce or eliminatethe ability of the polymerase to add non-templated 3′ nucleotides to asynthesized nucleic acid molecule.
 35. The polymerase of claim 34,wherein said polymerase is a DNA or RNA polymerase.
 36. The polymeraseof claim 34, wherein said polymerase is substantially pure.
 37. Thepolymerase of claim 34, wherein said polymerase is mesophilic orthermostable.
 38. The polymerase of claim 34, wherein said polymerase isselected from the group consisting of Tne DNA polymerase, Taq DNApolymerase, Tma DNA polymerase, Tth DNA polymerase, Tli DNA polymerase,VENT™ DNA polymerase, Pfu DNA polymerase, DEEPVENT™ DNA polymerase, PwoDNA polymerase, Bst DNA polymerase, Bca DNA polymerase, Tfl DNApolymerase, and mutants, variants and derivatives thereof.
 39. Thepolymerase of claim 34, wherein said polymerase is substantially reducedin at least one activity selected from the group consisting of: (a)3′→5′ exonuclease activity; and (b) 5′→3′ exonuclease activity.
 40. Thepolymerase of claim 39, wherein said polymerase is substantially reducedin 3′-5′ exonuclease activity.
 41. The polymerase of claim 39, whereinsaid polymerase is substantially reduced in 5′-3′ exonuclease activity.42. The polymerase of claim 41, which is modified or mutated to reduceor eliminate 3′-5′ exonuclease activity.
 43. The polymerase of claim 34,which comprises one or more modifications or mutations in the O-helix ofsaid polymerase.
 44. The polymerase of claim 43, wherein said O-helix isdefined as RXXXKXXXFXXXYX (SEQ ID NO:11), wherein X is any amino acid.45. The polymerase of claim 44, wherein said mutation or modification isat position R (Arg) and/or F (Phe) and/or K (Lys) of said O-helix orcombinations thereof.
 46. The polymerase of claim 44, wherein saidmutation or modification is an amino acid substitution at position Rand/or F and/or K of said O-helix or combinations thereof.
 47. Thepolymerase of claim 46, wherein R (Arg) is substituted with an aminoacid selected from the group consisting of Ala, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Try and Val. 49.The polymerase of claim 46, wherein F (Phe) is substituted with an aminoacid selected from the group consisting of Ala, Asn, Arg, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Try and Val. 50.The polymerase of claim 46, wherein K (Lys) is substituted with an aminoacid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Try and Val. 51.The polymerase of claim 46, wherein K (Lys) is substituted with Arg orHis.
 52. A mutant Tne DNA polymerase protein selected from the groupconsisting of: Tne N′Δ219, D323A; Tne N′Δ283, D323A; Tne N′Δ284, D323A;Tne N′Δ193, D323A; Tne D137A, D323A; Tne D8A, D323A; Tne G195D, D323A;Tne G37D, D323A; Tne N′Δ283; Tne D137A, D323A, R722K; Tne D137A, D323A,R722Y; Tne D137A, D323A, R722L; Tne D137A, D323A, R722H; Tne D137A,D323A, R722Q; Tne D137A, D323A, F730Y; Tne D137A, D323A, K726R; TneD137A, D323A, K726H; Tne D137A, D323A, R722K, F730Y; Tne D137A, D323A,R722K, K726R; Tne D137A, D323A, R722K, K726H; Tne D137A, D323A, R722H,F730Y; Tne D137A, D323A, R722H, K726R; Tne D137A, D323A, R722H, K726H;Tne D137A, D323A, R722Q, F730Y; Tne D137A, D323A, R722Q, K726R; TneD137A, D323A, R722Q, K726H; Tne D137A, D323A, R722N, F730Y; Tne D137A,D323A, R722N, K726R; Tne D137A, D323A, R722N, K726H; Tne D137A, D323A,F730S; Tne N′Δ283, D323A, R722K/H/Q/N/Y/L; Tne N′Δ219, D323A, R722K; TneN′Δ219, D323A, F730Y; Tne N′Δ219, D323A, K726R; Tne N′Δ219, D323A,K726H; Tne D137A, D323A, F730S, R722K/Y/Q/N/H/L, K726R/H; Tne D137A,D323A, F730T, R722K/Y/Q/N/H/L, K726R/H; Tne D137A, D323A, F730T; TneF730S; Tne F730A; Tne K726R; Tne K726H; and Tne D137A, D323A, R722N. 53.A vector comprising a gene encoding the polymerase of claim
 34. 54. Thevector of claim 53, wherein said gene is operably linked to a promoter.55. The vector of claim 54, wherein said promoter is selected from thegroup consisting of a λ-P_(L) promoter, a tac promoter, a trp promoter,and a trc promoter.
 56. A host cell comprising the vector of claim 53.57. A method of producing a polymerase, said method comprising: (a)culturing the host cell of claim 56; (b) expressing said gene; and (c)isolating said polymerase from said host cell.
 58. A method ofsynthesizing a nucleic acid molecule comprising: (a) mixing a nucleicacid template with one or more polymerases of claim 34; and (b)incubating said mixture under conditions sufficient to make a nucleicacid molecule complementary to all or a portion of said template. 59.The method of claim 58, wherein said mixture further comprises one ormore nucleotides selected from the group consisting of dATP, dCTP, dGTP,dTTP, dITP, 7-deaza-dGTP, dUTP, ddATP, ddCTP, ddGTP, ddlTP, ddTTP,[α-S]dATP, [α-S]dTTP, [α-S]dGTP, and [α-S]dCTP.
 60. The method of claim59, wherein one or more of said nucleotides are detectably labeled. 61.A method of sequencing a DNA molecule, comprising: (a) hybridizing aprimer to a first DNA molecule; (b) contacting said DNA molecule of step(a) with deoxyribonucleoside triphosphates, one or more DNA polymerasesof claim 34, and a terminator nucleotide; (c) incubating the mixture ofstep (b) under conditions sufficient to synthesize a random populationof DNA molecules complementary to said first DNA molecule, wherein saidsynthesized DNA molecules are shorter in length than said first DNAmolecule and wherein said synthesized DNA molecules comprise aterminator nucleotide at their 3′ termini; and (d) separating saidsynthesized DNA molecules by size so that at least a part of thenucleotide sequence of said first DNA molecule can be determined. 62.The method of claim 61, wherein said deoxyribonucleoside triphosphatesare selected from the group consisting of dATP, dCTP, dGTP, dTTP, dITP,7-deaza-dGTP, dUTP, [α-S]dATP, [α-S]dTTP, [α-S]dGTP, and [α-S]dCTP. 63.The method of claim 61, wherein said terminator nucleotide is ddTTP,ddATP, ddGTP, ddITP or ddCTP.
 64. The method of claim 61, wherein one ormore of said deoxyribonucleoside triphosphates is detectably labeled.65. The method of claim 61, wherein one or more of said terminatornucleotides is detectably labeled.
 66. A method for amplifying a doublestranded DNA molecule, comprising: (a) providing a first and secondprimer, wherein said first primer is complementary to a sequence at ornear the 3′-termini of the first strand of said DNA molecule and saidsecond primer is complementary to a sequence at or near the 3′-terminiof the second strand of said DNA molecule; (b) hybridizing said firstprimer to said first strand and said second primer to said second strandin the presence of the one or more DNA polymerases of claims 34, underconditions such that a third DNA molecule complementary to said firststrand and a fourth DNA molecule complementary to said second strand aresynthesized; (c) denaturing said first and third strand, and said secondand fourth strands; and (d) repeating steps (a) to (c) one or moretimes.
 67. A kit for sequencing, amplifying or sequencing a DNA moleculecomprising one or more polymerases of claim
 34. 68. The kit of claim 67,further comprising one or more dideoxyribonucleoside triphosphatesand/or one or more deoxyribonucleoside triphosphates.